Ptr1 evolved convergently with RPS2 and Mr5 to mediate recognition of AvrRpt2 in diverse solanaceous species.

The Ptr1 (Pseudomonas tomato race 1) locus in Solanum lycopersicoides confers resistance to strains of Pseudomonas syringae pv. tomato expressing AvrRpt2 and Ralstonia pseudosolanacearum expressing RipBN. Here we describe the identification and phylogenetic analysis of the Ptr1 gene. A single recombinant among 585 F2 plants segregating for the Ptr1 locus was discovered that narrowed the Ptr1 candidates to eight nucleotide-binding leucine-rich repeat protein (NLR)-encoding genes. From analysis of the gene models in the S. lycopersicoides genome sequence and RNA-Seq data, two of the eight genes emerged as the strongest candidates for Ptr1. One of these two candidates was found to encode Ptr1 based on its ability to mediate recognition of AvrRpt2 and RipBN when it was transiently expressed with these effectors in leaves of Nicotiana glutinosa. The ortholog of Ptr1 in tomato and in Solanum pennellii is a pseudogene. However, a functional Ptr1 ortholog exists in Nicotiana benthamiana and potato, and both mediate recognition of AvrRpt2 and RipBN. In apple and Arabidopsis, recognition of AvrRpt2 is mediated by the Mr5 and RPS2 proteins, respectively. Phylogenetic analysis places Ptr1 in a distinct clade compared with Mr5 and RPS2, and it therefore appears to have arisen by convergent evolution for recognition of AvrRpt2.

Survey of Sensitivity to Fatty Acid-Amino Acid Conjugates in the Solanaceae.

Plants perceive insect herbivores via a sophisticated surveillance system that detects a range of alarm signals, including herbivore-associated molecular patterns (HAMPs). Fatty acid-amino acid conjugates (FACs) are HAMPs present in oral secretions (OS) of lepidopteran larvae that induce defense responses in many plant species. In contrast to eggplant (Solanum melongena), tomato (S. lycopersicum) does not respond to FACs present in OS from Manduca sexta (Lepidoptera). Since both plants are found in the same genus, we tested whether loss of sensitivity to FACs in tomato may be a domestication effect. Using highly sensitive MAP kinase (MAPK) phosphorylation assays, we demonstrate that four wild tomato species and the closely related potato (S. tuberosum) do not respond to the FACs N-linolenoyl-L-glutamine and N-linolenoyl-L-glutamic acid, excluding a domestication effect. Among other genera within the Solanaceae, we found that bell pepper (Capsicum annuum) is responsive to FACs, while there is a differential responsiveness to FACs among tobacco (Nicotiana) species, ranging from strong responsiveness in N. benthamiana to no responsiveness in N. knightiana. The Petunia lineage is one of the oldest lineages within the Solanaceae and P. hybrida was responsive to FACs. Collectively, we demonstrate that plant responsiveness to FACs does not follow simple phylogenetic relationships in the family Solanaceae. Instead, sensitivity to FACs is a dynamic ancestral trait present in monocots and eudicots that was repeatedly lost during the evolution of Solanaceae species. Although tomato is insensitive to FACs, we found that other unidentified factors in M. sexta OS induce defenses in tomato.

A novel R3 MYB transcriptional repressor associated with the loss of floral pigmentation in Iochroma.

Losses of floral pigmentation represent one of the most common evolutionary transitions in flower color, yet the genetic basis for these changes has been elucidated in only a handful of cases. Here we used crossing studies, bulk-segregant RNA sequencing, phylogenetic analyses and functional tests to identify the gene(s) responsible for the transition to white flowers in Iochroma loxense. Crosses between I. loxense and its blue-flowered sister species, I. cyaneum, suggested that a single locus controls the flower color difference and that the white allele causes a nearly complete loss of pigmentation. Examining sequence variation across phenotypic pools from the crosses, we found that alleles at a novel R3 MYB transcription factor were tightly associated with flower color variation. This gene, which we term MYBL1, falls into a class of MYB transcriptional repressors and, accordingly, higher expression of this gene is associated with downregulation of multiple anthocyanin pigment pathway genes. We confirmed the repressive function of MYBL1 through stable transformation of Nicotiana. The mechanism underlying the evolution of white flowers in I. loxense differs from that uncovered in previous studies, pointing to multiple mechanisms for achieving fixed transitions in flower color intensity.

Solanaceae plant malformation in Chongqing City, China, reveals a pollution threat to the Yangtze River.

Water quality is under increasing threat from industrial and natural sources of pollutants. Here, we present our findings about a pollution incident involving the tap water of Chongqing City in China. In recent years, Solanaceae plants grown in greenhouses in this city have displayed symptoms of cupped, strappy leaves. These symptoms resembled those caused by chlorinated auxinic herbicides. We have determined that these symptoms were caused by the tap water used for irrigation. Using a bioactivity-guided fractionation method, we isolated a substance with corresponding auxinic activity from the tap water. The substance was named "solanicide" because of its strong bioactivity against Solanaceae plants. Further investigation revealed that the solanicide in the water system of Chongqing City is derived from the Jialing River, a major tributary of the Yangtze River. Therefore, it is also present in the Yangtze River downstream of Chongqing after the inflow of the Jialing River. Biological analyses indicated that solanicide is functionally similar to, but distinct from, other known chlorinated auxinic herbicides. Chemical assays further showed that solanicide structurally differs from those compounds. This study has highlighted a water pollution threat to the Yangtze River and its floodplain ecosystem.

Identification of microRNAs in six solanaceous plants and their potential link with phosphate and mycorrhizal signaling.

To date, only a limited number of solanaceous miRNAs have been deposited in the miRNA database. Here, genome-wide bioinformatic identification of miRNAs was performed in six solanaceous plants (potato, tomato, tobacco, eggplant, pepper, and petunia). A total of 2,239 miRNAs were identified following a range of criteria, of which 982 were from potato, 496 from tomato, 655 from tobacco, 46 from eggplant, 45 were from pepper, and 15 from petunia. The sizes of miRNA families and miRNA precursor length differ in all the species. Accordingly, 620 targets were predicted, which could be functionally classified as transcription factors, metabolic enzymes, RNA and protein processing proteins, and other proteins for plant growth and development. We also showed evidence for miRNA clusters and sense and antisense miRNAs. Additionally, five Pi starvation- and one arbuscular mycorrhiza (AM)-related cis-elements were found widely distributed in the putative promoter regions of the miRNA genes. Selected miRNAs were classified into three groups based on the presence or absence of P1BS and MYCS cis-elements, and their expression in response to Pi starvation and AM symbiosis was validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). These results show that conserved miRNAs exist in solanaceous species and they might play pivotal roles in plant growth, development, and stress responses.

Plant mating system transitions drive the macroevolution of defense strategies.

Understanding the factors that shape macroevolutionary patterns in functional traits is a central goal of evolutionary biology. Alternative strategies of sexual reproduction (inbreeding vs. outcrossing) have divergent effects on population genetic structure and could thereby broadly influence trait evolution. However, the broader evolutionary consequences of mating system transitions remain poorly understood, with the exception of traits related to reproduction itself (e.g., pollination). Across a phylogeny of 56 wild species of Solanaceae (nightshades), we show here that the repeated, unidirectional transition from ancestral self-incompatibility (obligate outcrossing) to self-compatibility (increased inbreeding) leads to the evolution of an inducible (vs. constitutive) strategy of plant resistance to herbivores. We demonstrate that inducible and constitutive defense strategies represent evolutionary alternatives and that the magnitude of the resulting macroevolutionary tradeoff is dependent on the mating system. Loss of self-incompatibility is also associated with the evolution of increased specificity in induced plant resistance. We conclude that the evolution of sexual reproductive variation may have profound effects on plant-herbivore interactions, suggesting a new hypothesis for the evolution of two primary strategies of plant defense.

Plant hormones including ethylene are recruited in calyx inflation in Solanaceous plants.

Plant hormones direct many processes of floral and post-floral morphogenesis in Angiosperms. However, their role in shaping floral morphological novelties, such as inflated calyx syndrome (ICS) exhibited by a few genera of the Solanaceae, remains unknown. In Withania and Physalis, sepals resume growth after pollination and encapsulate the mature fruit to form a balloon-like structure, i.e. ICS. The epidermal cells of calyx show enlargement and lobation post-fertilization. Application of hormones to depistillated flower buds of Withania revealed that cytokinins and gibberellins mimic fertilization signals. The ICS development is a synchronous step with fruit development; both processes are under the control of more or less the same set of hormones, including cytokinins and gibberellic acids. Interestingly, inhibition of ethylene in the system is sufficient to yield inflated calyx in Withania. In contrast, Tubocapsicum, a closely related species and an evolutionary natural loss mutant of ICS - showed no response to applied hormones, and ethylene led to inflation of the receptacle indirectly. In addition to hormones, the expression of an MPF2-like MADS-box transcription factor in sepals is essential for ICS formation. Nevertheless, the interactions between MPF2-like genes and hormones are barely detectable at the transcript level. Our data provide insight into the role of hormones in generating floral morphological diversity during evolution.

Multihost experimental evolution of a plant RNA virus reveals local adaptation and host-specific mutations.

For multihost pathogens, adaptation to multiple hosts has important implications for both applied and basic research. At the applied level, it is one of the main factors determining the probability and the severity of emerging disease outbreaks. At the basic level, it is thought to be a key mechanism for the maintenance of genetic diversity both in host and pathogen species. Using Tobacco etch potyvirus (TEV) and four natural hosts, we have designed an evolution experiment whose strength and novelty are the use of complex multicellular host organism as hosts and a high level of replication of different evolutionary histories and lineages. A pattern of local adaptation, characterized by a higher infectivity and virulence on host(s) encountered during the experimental evolution was found. Local adaptation only had a cost in terms of performance on other hosts in some cases. We could not verify the existence of a cost for generalists, as expected to arise from antagonistic pleiotropy and other genetic mechanisms generating a fitness trade-off between hosts. This observation confirms that this classical theoretical prediction lacks empirical support. We discuss the reasons for this discrepancy between theory and experiment in the light of our results. The analysis of full genome consensus sequences of the evolved lineages established that all mutations shared between lineages were host specific. A low degree of parallel evolution was observed, possibly reflecting the various adaptive pathways available for TEV in each host. Altogether, these results reveal a strong adaptive potential of TEV to new hosts without severe evolutionary constraints.

Comparative evidence for the correlated evolution of polyploidy and self-compatibility in Solanaceae.

Breakdown of self-incompatibility occurs repeatedly in flowering plants with important evolutionary consequences. In plant families in which self-incompatibility is mediated by S-RNases, previous evidence suggests that polyploidy may often directly cause self-compatibility through the formation of diploid pollen grains. We use three approaches to examine relationships between self-incompatibility and ploidy. First, we test whether evolution of self-compatibility and polyploidy is correlated in the nightshade family (Solanaceae), and find the expected close association between polyploidy and self-compatibility. Second, we compare the rate of breakdown of self-incompatibility in the absence of polyploidy against the rate of breakdown that arises as a byproduct of polyploidization, and we find the former to be greater. Third, we apply a novel extension to these methods to show that the relative magnitudes of the macroevolutionary pathways leading to self-compatible polyploids are time dependent. Over small time intervals, the direct pathway from self-incompatible diploids is dominant, whereas the pathway through self-compatible diploids prevails over longer time scales. This pathway analysis is broadly applicable to models of character evolution in which sequential combinations of rates are compared. Finally, given the strong evidence for both irreversibility of the loss of self-incompatibility in the family and the significant association between self-compatibility and polyploidy, we argue that ancient polyploidy is highly unlikely to have occurred within the Solanaceae, contrary to previous claims based on genomic analyses.

RanGAP2 mediates nucleocytoplasmic partitioning of the NB-LRR immune receptor Rx in the Solanaceae, thereby dictating Rx function.

The potato (Solanum tuberosum) nucleotide binding-leucine-rich repeat immune receptor Rx confers resistance to Potato virus X (PVX) and requires Ran GTPase-activating protein 2 (RanGAP2) for effective immune signaling. Although Rx does not contain a discernible nuclear localization signal, the protein localizes to both the cytoplasm and nucleus in Nicotiana benthamiana. Transient coexpression of Rx and cytoplasmically localized RanGAP2 sequesters Rx in the cytoplasm. This relocation of the immune receptor appeared to be mediated by the physical interaction between Rx and RanGAP2 and was independent of the concomitant increased GAP activity. Coexpression with RanGAP2 also potentiates Rx-mediated immune signaling, leading to a hypersensitive response (HR) and enhanced resistance to PVX. Besides sequestration, RanGAP2 also stabilizes Rx, a process that likely contributes to enhanced defense signaling. Strikingly, coexpression of Rx with the Rx-interacting WPP domain of RanGAP2 fused to a nuclear localization signal leads to hyperaccumulation of both the WPP domain and Rx in the nucleus. As a consequence, both Rx-mediated resistance to PVX and the HR induced by auto-active Rx mutants are significantly suppressed. These data show that a balanced nucleocytoplasmic partitioning of Rx is required for proper regulation of defense signaling. Furthermore, our data indicate that RanGAP2 regulates this partitioning by serving as a cytoplasmic retention factor for Rx.

On 'various contrivances': pollination, phylogeny and flower form in the Solanaceae.

Members of the euasterid angiosperm family Solanaceae have been characterized as remarkably diverse in terms of flower morphology and pollinator type. In order to test the relative contribution of phylogeny to the pattern of distribution of floral characters related to pollination, flower form and pollinators have been mapped onto a molecular phylogeny of the family. Bilateral flower symmetry (zygomorphy) is prevalent in the basal grades of the family, and more derived clades have flowers that are largely radially symmetric, with some parallel evolution of floral bilateralism. Pollinator types ('syndromes') are extremely homoplastic in the family, but members of subfamily Solanoideae are exceptional in being largely bee pollinated. Pollinator relationships in those genera where they have been investigated more fully are not as specific as flower morphology and the classical pollinator syndrome models might suggest, and more detailed studies in some particularly variable genera, such as Iochroma and Nicotiana, are key to understanding the role of pollinators in floral evolution and adaptive radiation in the family. More studies of pollinators in the field are a priority.

Variation of pollinator assemblages and pollen limitation in a locally specialized system: the oil-producing Nierembergia linariifolia (Solanaceae).

BACKGROUND AND AIMS: Few studies have examined the dynamics of specialist plant-pollinator interactions at a geographical scale. This knowledge is crucial for a more general evolutionary and ecological understanding of specialized plant-pollinator systems. In the present study, variations in pollinator activity, assemblage composition and pollen limitation were explored in the oil-producing species Nierembergia linariifolia (Solanaceae). METHODS: Pollen limitation in fruit and seed production was analysed by supplementary hand pollination in five wild populations. Pollinator activity and identity were recorded while carrying out supplementary pollination to assess the effect of pollinators on the degree of pollen limitation. In two populations, pollen limitation was discriminated into quantitative and qualitative components by comparing supplementation and hand cross-pollination in fruit set and seed set. The effect of flower number per plant on the number of flowers pollinated per visitor per visit to a plant was examined in one of these populations as a possible cause of low-quality pollination by increasing geitonogamy. RESULTS AND CONCLUSIONS: Although pollen limitation was evident along time and space, differences in magnitude were detected among populations and years that were greatly explained by pollinator activity, which was significantly different across populations. Floral display size had a significant effect on the visitation rate per flower. Limitation by quality clearly affected one population presumably due to a high proportion of geitonogamous pollen. The great inter-population variation in plant-pollinator interaction (both in pollinator assemblages composition and pollinator activity) and fitness consequences, suggests that this system should be viewed as a mosaic of locally selective processes and locally specialized interactions.

A Pseudomonas syringae pv. tomato DC3000 mutant lacking the type III effector HopQ1-1 is able to cause disease in the model plant Nicotiana benthamiana.

The model pathogen Pseudomonas syringae pv. tomato DC3000 causes bacterial speck in tomato and Arabidopsis, but Nicotiana benthamiana, an important model plant, is considered to be a non-host. Strain DC3000 injects approximately 28 effector proteins into plant cells via the type III secretion system (T3SS). These proteins were individually delivered into N. benthamiana leaf cells via T3SS-proficient Pseudomonas fluorescens, and eight, including HopQ1-1, showed some capacity to cause cell death in this test. Four gene clusters encoding 13 effectors were deleted from DC3000: cluster II (hopH1, hopC1), IV (hopD1, hopQ1-1, hopR1), IX (hopAA1-2, hopV1, hopAO1, hopG1), and native plasmid pDC3000A (hopAM1-2, hopX1, hopO1-1, hopT1-1). DC3000 mutants deleted for cluster IV or just hopQ1-1 acquired the ability to grow to high levels and produce bacterial speck lesions in N. benthamiana. HopQ1-1 showed other hallmarks of an avirulence determinant in N. benthamiana: expression in the tobacco wildfire pathogen P. syringae pv. tabaci 11528 rendered this strain avirulent in N. benthamiana, and elicitation of the hypersensitive response in N. benthamiana by HopQ1-1 was dependent on SGT1. DC3000 polymutants involving other effector gene clusters in a hopQ1-1-deficient background revealed that clusters II and IX contributed to the severity of lesion symptoms in N. benthamiana, as well as in Arabidopsis and tomato. The results support the hypothesis that the host ranges of P. syringae pathovars are limited by the complex interactions of effector repertoires with plant anti-effector surveillance systems, and they demonstrate that N. benthamiana can be a useful model host for DC3000.

Structure and function of resistance proteins in solanaceous plants.

Gene-for-gene resistance in plants is based on the presence of a resistance (R) gene in the host and a matching Avirulence (Avr) gene in the pathogen. Many R genes have been cloned over the past two decades, mostly from the Solanaceae. The gene products, called R proteins, display modular domain structures. R protein function has recently been shown to require dynamic interactions between the various domains. In addition to these intramolecular interactions, R proteins interact with other proteins to form signaling complexes that are able to activate an innate immune response that arrests proliferation of the invading pathogen, thereby conferring disease resistance. In this review, we summarize current understanding of R protein structure and function, as well as the molecular mechanisms underlying the activation of defense signaling processes. As well as being a rich source for R genes, Solanaceae are a leading model system in which to study inter- and intramolecular interactions of R proteins.

Conservation and divergence of both phosphate- and mycorrhiza-regulated physiological responses and expression patterns of phosphate transporters in solanaceous species.

Here, orthologous genes of six phosphate transporter (PiT) genes, which are members of the Pht1 and Pht2 families in tomato and potato, have been cloned from the solanaceous species pepper, eggplant and tobacco. Overall, expressions of these genes in pepper, eggplant and tobacco showed similar patterns to those in tomato and potato: P-starvation enhancement in both leaves and roots for Pht1;1, P-depletion induction exclusively in roots for Pht1;2, mycorrhizal enhancement for Pht1;3, and mycorrhizal induction for both Pht1;4 and Pht1;5. In the roots of nonmycorrhizal eggplant, SmPht1;3, SmPht1;4 and SmPht1;5 were also expressed under extreme P starvation. Mycorrhizal symbiosis under high-P supply conditions reduced plant growth, with concurrent enhancement of Pht1;2 expression in the roots of pepper as well as eggplant. In addition, the mycorrhizal symbiosis down-regulated the expression of Pht2;1 genes greatly in the leaves of pepper and tobacco. The discrepancies between the evolutionary distances of the PiT genes and their expression patterns among the five species suggest greater complexity in function of PiT in plants than previously expected.

Systemins: a functionally defined family of peptide signals that regulate defensive genes in Solanaceae species.

Numerous plant species have been known for decades that respond to herbivore attacks by systemically synthesizing defensive chemicals to protect themselves from predators. The nature of systemic wound signals remained obscure until 1991, when an 18-aa peptide called systemin was isolated from tomato leaves and shown to be a primary signal for systemic defense. More recently, two new hydroxyproline-rich, glycosylated peptide defense signals have been isolated from tobacco leaves, and three from tomato leaves. Because of their origins in plants, small sizes, hydroxyproline contents (tomato systemin is proline-rich), and defense-signaling activities, the new peptides are included in a functionally defined family of signals collectively called systemins. Here, we review structural and biological properties of the systemin family, and discuss their possible roles in systemic wound signaling.

S-RNase-mediated self-incompatibility.

The Solanaceae, Rosaceae, and Scrophulariaceae families all possess an RNase-mediated self-incompatibility mechanism through which their pistils can recognize and reject self-pollen to prevent inbreeding. The highly polymorphic S-locus controls the self-incompatibility interaction, and the S-locus of the Solanaceae has been shown to be a multi-gene complex in excess of 1.3 Mb. To date, the function of only one of the S-locus genes, the S-RNase gene, has been determined. This article reviews the current status of the search for the pollen S-gene and the current models for how S-haplotype specific inhibition of pollen tubes can be accomplished by S-RNases.

A pentatricopeptide repeat-containing gene restores fertility to cytoplasmic male-sterile plants.

Known in over 150 species, cytoplasmic male sterility is encoded by aberrant mitochondrial genes that prevent pollen development. The RNA- or protein-level expression of most of the mitochondrial genes encoding cytoplasmic male sterility is altered in the presence of one or more nuclear genes called restorers of fertility that suppress the male-sterile phenotype. Cytoplasmic male sterility/restorer systems have been proven to be an invaluable tool in the production of hybrid seeds. Despite their importance for both the production of major crops such as rice and sunflower and the study of organelle/nuclear interactions in plants, none of the nuclear fertility-restorer genes that reduce the expression of aberrant mitochondrial proteins have previously been cloned. Here we report the isolation of a gene directly involved in the control of the expression of a cytoplasmic male sterility-encoding gene. The Petunia restorer of fertility gene product is a mitochondrially targeted protein that is almost entirely composed of 14 repeats of the 35-aa pentatricopeptide repeat motif. In a nonrestoring genotype we identified a homologous gene that exhibits a deletion in the promoter region and is expressed in roots but not in floral buds.

Extracellular freezing in leaves of freezing-sensitive species.

Low-temperature scanning-electron microscopy was used to study the freezing of leaves of five species that have no resistance to freezing: bean (Phaseolus vulgaris L.), tobacco (Nicotiana tabacum L.), tomato (Lycopersicon esculentum L.), cucumber (Cucumis sativus L.), and corn (Zea mays L.). In the leaves of the four dicotyledonous species, ice was extracellular and the cells of all tissues were collapsed. In contrast, in maize leaves ice was extracellular in the mesophyll, and these cells were collapsed, but the epidermal and bundle-sheath cells apparently retained their original shapes and volume. It is concluded that the leaves of the freezing-sensitive dicotyledonous species tested were killed by cellular dehydration induced by extracellular freezing, and not by intracellular freezing. Freezing injury in maize leaves apparently resulted from a combination of freezing-induced cellular dehydration of some cells and intracellular ice formation in epidermal and bundle-sheath cells.