Literatura académica sobre el tema ""nor tomato mutant""

Abstract Differences in aroma have been examined in headspace samples of ripe tomato (Lycopersicon esculentum Mill.) fruit of ‘Rutgers’ and of yellowing fruit of the nonripening mutants rin and nor. Volatiles were trapped and separated by gas chromatography, and the intensity of the effluent aromas was rated by sniffing. Intense aroma compounds were identified by mass spectrometry. Sixty-nine intense compounds were found in ‘Rutgers’, of which 46 were present in one or both mutant strains. Fifteen compounds with odor intensities rated medium to very strong were identified that were deficient or absent in fruit of the mutants. The latter compounds included two aldehydes, seven alcohols, two ketones, three sulfur-containing compounds, and a phenol. A few compounds were intense odors in ‘Rutgers’ and in one or both mutants; hex-2-enal, linalool, phenylacetaldehyde, methyl salicylate, 2-phenylethanol, and eugenol. Some compounds were detected that were more intense in rin and nor than in ‘Rutgers’ (e.g., guaiacol). It is proposed that the “normal background aroma” in fresh tomatoes is caused by those intense odors, which are common to both normal and mutant strains, whereas the bland flavor of mutant fruit is caused by the absence of those intense aroma compounds found only in ‘Rutgers’. The intense aroma compounds found only in ‘Rutgers’ may be crucial determinants of acceptability in fresh tomato fruit.

Abstract The tomato non-ripening (nor) mutant generates a truncated 186-amino-acid protein (NOR186) and has been demonstrated previously to be a gain-of-function mutant. Here, we provide more evidence to support this view and answer the open question of whether the NAC-NOR gene is important in fruit ripening. Overexpression of NAC-NOR in the nor mutant did not restore the full ripening phenotype. Further analysis showed that the truncated NOR186 protein is located in the nucleus and binds to but does not activate the promoters of 1-aminocyclopropane-1-carboxylic acid synthase2 (SlACS2), geranylgeranyl diphosphate synthase2 (SlGgpps2), and pectate lyase (SlPL), which are involved in ethylene biosynthesis, carotenoid accumulation, and fruit softening, respectively. The activation of the promoters by the wild-type NOR protein can be inhibited by the mutant NOR186 protein. On the other hand, ethylene synthesis, carotenoid accumulation, and fruit softening were significantly inhibited in CR-NOR (CRISPR/Cas9-edited NAC-NOR) fruit compared with the wild-type, but much less severely affected than in the nor mutant, while they were accelerated in OE-NOR (overexpressed NAC-NOR) fruit. These data further indicated that nor is a gain-of-function mutation and NAC-NOR plays a significant role in ripening of wild-type fruit.

Abstract Fungal cell-wall lysing enzymes have been shown to induce ethylene production in different plant systems. The effect of endogenous plant cell-wall lysing enzymes on ethylene synthesis in fruit has received only limited attention. Therefore, tomato fruit (Lycopersicon esculentum, Mill.) were vacuum–infiltrated with the tomato cell-wall enzymes, polygalacturonase I and II (PG I, PG II) and pectinmethylesterase (PME). Fruit ethylene levels were observed to increase relative to either salt, buffer, or boiled enzyme controls. This increase in ethylene production occurred in green ‘Cherry’ tomato fruit as well as in the mutants rin, nor, and Cornell 111. Enzyme-induced ethylene synthesis generally peaked at or before 17 to 20 hr and decreased to lower or basal levels in most immature normal cultivars by 42 hr after treatment. Ethylene was maintained at high levels, however, in some (possibly more mature) green fruit, as well as in all mutant lines. PG II was more effective than PG I in inducing ethylene production and PME seemed to enhance the ethylene-inducing activity of PG II.

The insertion site of a transposon mutant of Clavibacter michiganensis subsp. michiganensis NCPPB382 was cloned and found to be located in the gene tomA encoding a member of the glycosyl hydrolase family 10. The intact gene was obtained from a cosmid library of C. michiganensis subsp. michiganensis. The deduced protein TomA (543 amino acids, 58 kDa) contains a predicted signal peptide and two domains, the N-terminal catalytic domain and a C-terminal fibronectin III-like domain. The closest well-characterized relatives of TomA were tomatinases from fungi involved in the detoxification of the tomato saponin α-tomatine which acts as a growth inhibitor. Growth inhibition of C. michiganensis subsp. michiganensis by α-tomatine was stronger in the tomA mutants than in the wild type. Tomatinase activity assayed by deglycosylation of α-tomatine to tomatidine was demonstrated in concentrated culture supernatants of C. michiganensis subsp. michiganensis. No activity was found with the tomA mutants. However, neither the transposon mutant nor a second mutant constructed by gene disruption was affected in virulence on the tomato cv. Moneymaker.

Recent advances in molecular genetics and genomics technologies have had a significant impact on tomato research over the last decade and are likely to have considerable influence on the nature and outcome of research activities related to tomato in the future. Specific applications of genomics technologies in our laboratory include positional cloning of genes associated with fruit ripening and quality (rin and nor), localization of ripening-related genes on the molecular-marker map to assist candidate gene discovery related to fruit ripening and quality, and characterization of mutants influencing fruit quality and nutritional value with the goal of identifying candidate genes for said mutants and alternative molecular tools for modification of fruit quality and nutrition. Isolation of the rin and nor genes has been verified via complementation of corresponding mutant tomato plants via insertion of the appropriate CaMV35s-driven wild-type sense cDNA. Both the rin and nor genes have sequence characteristics suggestive of transcription factors. Preliminary evidence suggests the role of similar genes in the ripening of additional climacteric and non-climacteric fruit species. Additional efforts in the laboratory include molecular analyses of light signal transduction as related to 1) regulation of carotenoid and flavonoid accumulation, and 2) potential manipulation of corresponding pathways for modification of fruit quality and nutrient value.

Abstract: The objective of this work was to evaluate the effects of the mutant alleles alcobaça (nor A ), ripening inhibitor (rin), and old gold crimson (og c ), in heterozygosity or homozygosity, on the expression of color and on the postharvest quality of fruit of experimental tomato hybrids. Fourteen hybrids with contrasting genotypic constitutions in the nor A , rin, and og c loci were evaluated in a randomized complete block design with four replicates. The following fruit postharvest quality traits were evaluated: firmness in the breaker stage, color, and soluble solids content. The rin +/rin and nor +/nor A genotypes increased firmness of tomato fruit at harvest (breaker stage). The rin + /rin genotypes displayed the worse internal fruit color. There was a positive effect of og c+ /og c in improving the internal color of rin + /rin and nor + /nor A fruit, making the color similar to that of the normal genotypes. The combination of the og c /og c rin + /rin nor + /nor A genes is effective to improve tomato fruit firmness, besides maintaining or improving internal color.

Two tomato lines with normal maturation (NR-1 and NR-2) have been crossed with two mutant lines (NR-10 nor and NR-12 rin) with delayed maturation (shelf life). Determination of mutant genes has been done by ?2 test on 100 fruits from F2 generation. Fruits have been picked 65 days from antesis and kept for 60 days, when six evaluations have been done. Data have been collected every 10 days on parental lines and progeny F1 and F2 generation. Variance testing has been done on the basis of one- and two-factorial analysis and groups compared by contrasts. Fruits have been preserved in controlled conditions (in dark at 5?C). Tomato genotypes with nor or rin gene had desirable traits (delayed ripening, long shelf life and firm fruits) for modern selection, so they should be included in programmes aiming to create commercial F1 hybrids.

Bacterial speck of tomato, caused by Pseudomonas syringae pv. tomato, continues to be a problem for tomato growers worldwide. A collection of nonpathogenic bacteria from tomato leaves plus P. syringae strains TLP2 and Cit7, P. fluorescens strain A506, and P. syringae pv. tomato DC3000 hrp mutants were examined in a greenhouse bioassay for the ability to reduce foliar bacterial speck disease severity. While several of these strains significantly reduced disease severity, P. syringae Cit7 was the most effective, providing a mean level of disease reduction of 78% under greenhouse conditions. The P. syringae pv. tomato DC3000 hrpA, hrpH, and hrpS mutants also significantly reduced speck severity under greenhouse conditions. The strains with the greatest efficacy under greenhouse conditions were tested for the ability to reduce bacterial speck under field conditions at locations in Alabama, Florida, and Ontario, Canada. P. syringae Cit7 was the most effective strain, providing a mean level of disease reduction of 28% over 10 different field experiments. P. fluorescens A506, which is commercially available as Blight-Ban A506, provided a mean level of disease reduction of 18% over nine different field experiments. While neither P. syringae Cit7 nor P. fluorescens A506 can be integrated with copper bactericides due to their copper sensitivity, there exist some potential for integrating these biological control agents with “plant activators”, including Actigard. Of the P. syringae pv. tomato DC3000 hrp mutants tested, only the hrpS mutant reduced speck severity significantly under field conditions.

Responses of resistant (Mi-1/Mi-1) and susceptible (mi-1/ mi-1) tomato (Solanum lycopersicum) to root-knot nematodes (RKNs; Meloidogyne spp.) infection were monitored using cDNA microarrays, and the roles of salicylic acid (SA) and jasmonic acid (JA) defense signaling were evaluated in these interactions. Array analysis was used to compare transcript profiles in incompatible and compatible interactions of tomato roots 24 h after RKN infestation. The jai1 and def1 tomato mutant, altered in JA signaling, and tomato transgenic line NahG, altered in SA signaling, in the presence or absence of the RKN resistance gene Mi-1, were evaluated. The array analysis identified 1,497 and 750 genes differentially regulated in the incompatible and compatible interactions, respectively. Of the differentially regulated genes, 37% were specific to the incompatible interactions. NahG affected neither Mi-1 resistance nor basal defenses to RKNs. However, jai1 reduced tomato susceptibility to RKNs while not affecting Mi-1 resistance. In contrast, the def1 mutant did not affect RKN susceptibility. These results indicate that JA-dependent signaling does not play a role in Mi-1-mediated defense; however, an intact JA signaling pathway is required for tomato susceptibility to RKNs. In addition, low levels of SA might be sufficient for basal and Mi-1 resistance to RKNs.

The tomato is a research model for fruit-ripening, however, its fruit-ripening mechanism still needs more extensive and in-depth exploration. Here, using TMT and LC-MS, the proteome and phosphoproteome of AC++ (wild type) and rin (ripening-inhibitor) mutant fruits were studied to investigate the translation and post-translational regulation mechanisms of tomato fruit-ripening. A total of 6141 proteins and 4011 phosphorylation sites contained quantitative information. One-hundred proteins were identified in both omics’ profiles, which were mainly found in ethylene biosynthesis and signal transduction, photosynthesis regulation, carotenoid and flavonoid biosynthesis, chlorophyll degradation, ribosomal subunit expression changes, MAPK pathway, transcription factors and kinases. The affected protein levels were correlated with their corresponding gene transcript levels, such as NAC-NOR, MADS-RIN, IMA, TAGL1, MADS-MC and TDR4. Changes in the phosphorylation levels of NAC-NOR and IMA were involved in the regulation of tomato fruit-ripening. Although photosynthesis was inhibited, there were diverse primary and secondary metabolic pathways, such as glycolysis, fatty acid metabolism, vitamin metabolism and isoprenoid biosynthesis, regulated by phosphorylation. These data constitute a map of protein—protein phosphorylation in the regulation of tomato fruit-ripening, which lays the foundation for future in-depth study of the sophisticated molecular mechanisms of fruit-ripening and provide guidance for molecular breeding.

La vite (Vitis vinifera L.), una delle più coltivate piante da frutto, riveste notevole importanza economica in tutto il mondo. Poichè negli ultimi decenni la viticoltura sta subendo gli effetti del riscaldamento globale (Webb et al., 2007), è necessario mantenere una produzione di uva e vino di elevata qualità. Una delle maggiori sfide consiste nell’identificazione dei principali geni regolatori dello sviluppo della pianta di vite durante il ciclo vegetale annuale e, in particolare, della transizione dalla fase vegetativa a quella matura (detta véraison), durante la quale avvengono profonde modificazioni biochimiche, fisiologiche e trascrizionali. Grazie ad un'analisi di network di co-espressione sull’atlante del trascrittoma della vite e ad un dataset di dati trascrizionali di bacche (Massonnet, 2015; Palumbo et al., 2014; Fasoli et al., 2012), è stata identificata una nuova categoria di geni chiamata 'switch’; tali geni sono significativamente up-regolati durante la transizione di fase ed inversamente correlati a molti geni soppressi durante la fase matura. Tra questi, i fattori di trascrizione NAM/ATAF/CUC (NAC) rappresentano un’interessante famiglia genica dato il ruolo chiave in processi biologici come sviluppo e risposte allo stress in pianta (Jensen et al., 2014). Per la caratterizzazione funzionale cinque geni NAC sono stati selezionati come putativi principali regolatori della riprogrammazione del trascrittoma durante la maturazione della vite. VvNAC11, VvNAC13, VvNAC33 e VvNAC60 sono stati identificati come geni 'switch' dalla sopra citata analisi, mentre VvNAC03 come gene omologo a NOR (non-ripening) di pomodoro, uno dei principali regolatori della maturazione di tale frutto (Giovannoni, 2004; Giovannoni et al., 1995). I cinque NAC sono stati sovra-espressi transientemente in Vitis vinifera per ottenere una panoramica dei loro effetti primari sul trascrittoma. Sono poi state ottenute e caratterizzate dal punto di vista molecolare e fenotipico piante di vite stabilmente trasformate con VvNAC33 e VvNAC60. VvNAC33 sembra essere coinvolto nella regolazione negativa della fotosintesi poiché le foglie sovra-esprimenti tale gene contengono una minor quantità di clorofilla, mentre VvNAC60 provoca una ridotta crescita della pianta e una prematura lignificazione dello stelo rispetto ad una pianta controllo della stessa età. Questi risultati riflettono comportamenti tipici di piante in fase di maturazione e senescenza, sostenendo l’ipotesi di un ruolo fondamentale dei NAC nella transizione di fase in vite. Al fine di identificare i target che agiscono a valle dei NAC, sono state eseguite analisi microarray sulle foglie delle piante trasformate in modo transiente e stabile. In entrambe le over-espressioni è stata influenzata l’espressione di un'ampia gamma di processi cellulari tra cui, tra le categorie funzionali più rappresentate, vi sono trasporto, metabolismo secondario e attività dei fattori di trascrizione. L'identificazione di VvMYBA1, un noto regolatore della biosintesi degli anotciani in vite (Kobayashi et al., 2002), come target di VvNAC60 suggerisce un ruolo di tale NAC in processi tipici dell’inizio della maturazione. Un altro approccio utilizzato in questo lavoro è stato la complementazione funzionale del mutante nor di pomodoro con i NAC selezionati. Risultati preliminari hanno rivelato che VvNAC03 e VvNAC60 sembrano avere una funzione simile a NOR poichè riescono a maturare almeno esternamente. In conclusione, i risultati ottenuti in questo lavoro suggeriscono la capacità dei VvNAC selezionati di influenzare l'espressione di geni coinvolti nella regolazione che controlla lo sviluppo dalla fase vegeativa alla fase matura in vite. Questo lavoro ha inizato a far luce sul ruolo dei NAC nello sviluppo della vite, ma dovranno essere effettuate ulteriori analisi per ottenere una piena compresione del macchinario molecolare che regola questo complesso sistema di regolazione.
Grapevine is the most widely cultivated and economically important fruit crop in the world. Viticulture has been affected by the global warming currently under way over the past few decades (Webb et al., 2007). Improving the genetics of key grapevine functions is needed to keep producing high quality grapes and wine. In this context, a challenging task is to identify master regulators that program the development of grapevine organs and control transition from vegetative-to-mature growth featured by grape berries during the annual plant cycle. This transition, called véraison, is marked by profound biochemical, physiological and transcriptomic modifications that allow vegetative green berries to enter the ripening process. Thanks to an integrated network analysis performed on the grapevine global gene expression atlas and from a large berry transcriptomic data set (Massonnet, 2015; Palumbo et al., 2014; Fasoli et al., 2012) a new category of genes, called ‘switch’ genes, was identified; they were significantly up-regulated during the developmental shift and inversely correlated with many genes suppressed during the mature growth phase. Among them, plant-specific NAM/ATAF/CUC (NAC) transcription factors represent an interesting gene family due to their key role in the biological processes in plant development and stress responses (Jensen et al., 2014). Five NAC genes were selected for functional characterization as key factor candidates of the major transcriptome reprogramming during grapevine development. VvNAC11, VvNAC13, VvNAC33 and VvNAC60 were identified as ‘switch’ genes in the above-mentioned analysis whereas VvNAC03 was selected because it is a close homologue of tomato NOR (non-ripening), known for its crucial role in tomato fruit ripening regulation (Giovannoni, 2004; Giovannoni et al., 1995). Firstly, the five transcription factors were transiently over-expressed in Vitis vinifera to get an overview of their primary effects on native species. Secondly, we obtained grapevine plants that were stably transformed with VvNAC33 and VvNAC60 and subjected to molecular/phenotypic characterizations. VvNAC33 seemed to be involved in negative regulation of photosynthesis since over-expressing leaves revealed a chlorophyll breakdown, while VvNAC60 affected regular plant development, showing a slight growth and earlier stem lignification in comparison to a same-age plant control. These results reflected typical behaviors of plants undergoing ripening and senescence, thus supporting our working hypothesis proposing a crucial role of NACs in the transition from vegetative to mature development in grapevine. In order to identify downstream targets of the NAC transcription factors analyzed in this work, we performed microarray analysis on leaves of transient and stable ectopic expressing plants. We noted that both over-expressions affected a wide range of cellular processes and among the most represented functional categories we found transport, secondary metabolism and transcription factor activity. The identification of VvMYBA1, a known grapevine regulator of the anthocyanin biosynthetic pathway (Kobayashi et al., 2002), as VvNAC60 target suggests a VvNAC60 role in processes like anthocyanin biosynthesis featured by grape berries at the onset of ripening. Another approach used to clarify NACs roles was to check the ability of VvNACs to fulfil the tomato NOR function. Preliminary results revealed that VvNAC03 and VvNAC60 could partially complement the nor mutation in tomato, establishing a partial ripening phenotype in fruits. Taken together, these findings suggest the ability of the selected VvNACs to affect the expression of genes involved in the regulatory network that controls the developmental shift to a mature phase in grapevine. This work has shed some light on the roles of these NACs in grapevine development, but further analysis must be conducted to fully elucidate the molecular machinery in this complex regulation system.

The intensity of root colonization by phytopathogenic fungus and rhizobacterium differs depending on the tomato genotype. Inoculation of wild-type tomatoes Ailsa Craig, but not of its ABA deficient mutant flacca, with Novosphingobium sp. P6W inhibits root colonization by Fusarium oxysporum MF-G284.

Fruit deterioration is a consequence of a genetically-determined fruit ripening and senescence programs, in which developmental factors lead to a climacteric rise of ethylene production in ethylene-sensitive fruits such as tomato and banana. Breeding of tomato with extended fruit shelf life involves the incorporation of a mutation in RIN, a MADS-box transcription factor participating in developmental control signalling of ripening. The RIN mode of action is not fully understood, and it may be predicted to interact with other MADS-box genes to execute its effects. The overall goal of this study was to demonstrate conservation of ripening control functions between banana and tomato and thus, the potential to genetically extend shelf-life in banana based on tools developed in tomato. The specific objectives were: 1. To increase the collection of potential RIN-like genes from banana; 2. To verify their action as developmental regulators; 3. To elucidate MADS-box gene mode of action in ripening control; 4. To create transgenic banana plants that express low levels of endogenous Le-RIN- like, MaMADS- gene(s). We have conducted experiments in banana as well as in tomato. In tomato we have carried out the transformation of the tomato rin mutant with the MaMADS1 and MaMADS2 banana genes. We have also developed a number of domain swap constructs to functionally examine the ripening-specific aspects of the RIN gene. Our results show the RIN-C terminal region is essential for the gene to function in the ripening signalling pathway. We have further explored the tomato genome databases and recovered an additional MADS-box gene necessary for fruit ripening. This gene has been previously termed TAGL1 but has not been functionally characterized in transgenic plants. TAGL1 is induced during ripening and we have shown via RNAi repression that it is necessary for both fleshy fruit expansion and subsequent ripening. In banana we have cloned the full length of six MaMADS box genes from banana and determined their spatial and temporal expression patterns. We have created antibodies to MaMADS2 and initiated ChI assay. We have created four types of transgenic banana plants designed to reduce the levels of two of the MaMADS box genes. Our results show that the MaMADS-box genes expression in banana is dynamically changing after harvest and most of them are induced at the onset of the climacteric peak. Most likely, different MaMADS box genes are active in the pulp and peel and they are differently affected by ethylene. Only the MaMADS2 box gene expression is not affected by ethylene indicating that this gene might act upstream to the ethylene response pathway. The complementation analysis in tomato revealed that neither MaMADS1 nor MaMADS2 complement the rin mutation suggesting that they have functionally diverged sufficiently to not be able to interact in the context of the tomato ripening regulatory machinery. The developmental signalling pathways controlling ripening in banana and tomato are not identical and/or have diverged through evolution. Nevertheless, at least the genes MaMADS1 and MaMADS2 constitute part of the developmental control of ripening in banana, since transgenic banana plants with reduced levels of these genes are delayed in ripening. The detailed effect on peel and pulp, of these transgenic plants is underway. So far, these transgenic bananas can respond to exogenous ethylene, and they seem to ripen normally. The response to ethylene suggest that in banana the developmental pathway of ripening is different than that in tomato, because rin tomatoes do not ripen in response to exogenous ethylene, although they harbor the ethylene response capability This study has a major contribution both in scientific and agricultural aspects. Scientifically, it establishes the role of MaMADS box genes in a different crop-the banana. The developmental ripening pathway in banana is similar, but yet different from that of the model plant tomato and one of the major differences is related to ethylene effect on this pathway in banana. In addition, we have shown that different components of the MaMADS-box genes are employed in peel and pulp. The transgenic banana plants created can help to further study the ripening control in banana. An important and practical outcome of this project is that we have created several banana transgenic plants with fruit of extended shelf life. These bananas clearly demonstrate the potential of MaMADS gene control for extending shelf-life, enhancing fruit quality, increasing yield in export systems and for improving food security in areas where Musaspecies are staple food crops.

The DNA of tomato yellow leaf curl virus (TYLCB) was detected in its whitefly vector, Bemisia tabaci, by dot spot hybridization as early as 1 h after acquisition access. The retention of the virus nucleic acid in the vector was at least 23 days after a 48 h acquisition access. However, the retention of TYLCV coat protein did not exceed 10 days. No replicative forms of TYLCV could be detected in B. tabaci, indicating a non-propagative relationship with the vector. Whiteflies were not able to accumulate naked virion ssDNA, virus cloned dsDNA, or virions with impaired coat protein. Deletion, frameshift, and single amino acid mutations were inserted into open reading frames (ORFs) V1 and V2 (Coat protein) of TYLCV. The ability of these mutants to replicate, to spread and to induce symptoms was tested both in leaf disks and in intact plants. No replication was found in tissues that were infected with a deletion mutant that lacked the carboxy half of the coat protein gene. Residual amounts of ssDNA and dsDNA were detected i tissues infected with a frameshift mutant in which an early termination at the extreme part of the protein. Two other mutants in which a single amino acid was changed in the overlapping part of V1 and V2 were able to spread systemically but infections remained symptomless and the production of ssDNA and dsDNA were significantly lower. These mutants were acquired and transmitted by Bemisia tabaci. Procedures for the the dissection, fixation and embedding of whiteflies were developed. The anatomy and ultrastructure of the salivary gland and the midgut of Bemisia tabaci and Trialeurodes vaporariorum (a vector and non-vector of geminiviruses respectively) was studied and described. Monoclonal antibodies against bean golden mosaic virus (BGMV) with narrow and broad spectrum were prepared. Transmission studies of tomato mottle geminivirus (TMoV) by B. tabaci were carried out. These studies were essential for a further work aimed to understand the interaction of geminiviruses with the insect and their localization in its tissues. To enable the production of transgenic plants procedures were developed for tomato transformation with both Agrobacterium and microparticle bombardment.

The primary objectives for the US lab included: the characterization of ELF3 transcription and translation; the creation and characterization of various transgenic lines that misexpress ELF3; defining genetic pathways related to ELF3 function regulating floral initiation in Arabidopsis; and the identification of genes that either interact with or are regulated by ELF3. Light quality, photoperiod, and temperature often act as important and, for some species, essential environmental cues for the initiation of flowering. However, there is relatively little information on the molecular mechanisms that directly regulate the developmental pathway from the reception of the inductive light signals to the onset of flowering and the initiation of floral meristems. The ELF3 gene was identified as possibly having a role in light-mediated floral regulation since elj3 mutants not only flower early, but exhibit light-dependent circadian defects. We began investigating ELF3's role in light signalling and flowering by cloning the ELF3 gene. ELF3 is a novel gene only present in plant species; however, there is an ELF3 homolog within Arabidopsis. The Arabidopsis elj3 mutation causes arrhythmic circadian output in continuous light; however, we show conclusively normal circadian function with no alteration of period length in elj3 mutants in dark conditions and that the light-dependent arrhythmia observed in elj3 mutants is pleiotropic on multiple outputs regardless of phase. Plants overexpressing ELF3 have an increased period length in constant light and flower late in long-days; furthermore, etiolated ELF3-overexpressing seedlings exhibit a decreased acute CAB2 response after a red light pulse, whereas the null mutant is hypersensitive to acute induction. This finding suggests that ELF3 negatively regulates light input to both the clock and its outputs. To determine whether ELF3's action is phase dependent, we examined clock resetting by light pulses and constructed phase response curves. Absence of ELF3 activity causes a significant alteration of the phase response curve during the subjective night, and overexpression of ELF3 results in decreased sensitivity to the resetting stimulus, suggesting that ELF3 antagonizes light input to the clock during the night. Indeed, the ELF3 protein interacts with the photoreceptor PHYB in the yeast two-hybrid assay and in vitro. The phase ofELF3 function correlates with its peak expression levels of transcript and protein in the subjective night. ELF3 action, therefore, represents a mechanism by which the oscillator modulates light resetting. Furthermore, flowering time is dependent upon proper expression ofELF3. Scientifically, we've made a big leap in the understanding of the circadian system and how it is coupled so tightly with light reception in terms of period length and clock resetting. Agriculturally, understanding more about the way in which the clock perceives and relays temporal information to pathways such as those involved in the floral transition can lead to increased crop yields by enabling plants to be grown in suboptimal conditions.

Hybrid vigor is the leading concept that rules crops breeding for almost a century. Yet, the exact mechanism that underlies heterosis is not clear. Over dominance interaction between alleles is one of the possible explanations. Our preliminary results indicated that severe developmental mutations at the heterozygous state have significant potential to improve plant performance. This led us to propose the ‘ODO breeding method’ that is based replacing a parental line of a successful hybrid with its mutated from to improve hybrid performance. Our BARD research challenged this method in three crop systems: maize, tomato and melon. In maize we could not detect any effect of mutant heterozigosity on yield or yield components when hybrids were tested however when we analyzed the effect of heterozigosity in mutant genes at B73 genetic background we could detect ODO in yield components using certain mutants. Our results indicate that the potential of utilizing the ODO Breeding Method for maize remains unclear. In tomato we overcame technical problems we faced in creating an EMS mutation library in indeterminate glasshouse tomatoes and now we have in our hands advanced material to study the putative ODO hybrids. We transferred some of the promising ODO mutations from M82 to indeterminate glasshouse tomatoes and putative ODO hybrids are ready to be evaluated this winter. In addition, we tested the effect of In melon we compared putative ‘ODO hybrids’ with their isogenic hybrids lacking the mutant allele and our results indicated a potential for the ODO breeding method to improve yield, fruit number per plant, and carotenoids content. Additional experiments are required to estimate better the expected success percentage of the ODO breeding method in melon so that it will become a recommended practice for improving hybrid performance. Based on our results we can't yet recommend the 'ODO breeding method' as a general tool to improve hybrid performance and more efforts are necessary to evaluate the percent of success of this method. The increased carotenoid content we found in association with CRTISO heterozygosity is promising and additional experiments are currently being performed to characterize this finding.

Fruit constitutes a major component of our diet, providing fiber, vitamins, minerals, and many other phytonutrients that promote good health. Fleshy fruits, such as tomatoes, already contain high levels of several of these ingredients. Nevertheless, efforts have been invested in increasing and diversifying the content of phytonutrients, such as carotenoids and flavonoids, in tomato fruits. Increasing levels of phytonutrients, such as lycopene, is highly justified from the perspective of the lycopene extraction industry due to cost effectiveness reasons. Diversifying phytonutrients, in particular those that contribute to fruit color, could potentially provide an array of attractive colors to our diet. Our major goal was to devise a novel strategy for developing tomato fruits with enhanced levels of phytochemicals known to promote good health with special emphasis on lycopene content. A further important goal was to analyze global gene expression of selected genetic lines produced throughout this study in order is to dissect the molecular mechanisms regulating phytonutrients accumulation in the tomato fruit. To achieve these goals we proposed to: 1. combine, by classical breeding, engineered polyamine metabolism with photomorphogenic high pigment mutants in order generate tomato plant with exceptionally high levels of phytonutrients; 2. use gene transfer technology for genetic introduction of key genes that promote phytonutrient accumulation in the tomato fruit, 3. Analyze accumulation patterns of the phytonutrients in the tomato fruit during ripening; 4. Analyze global gene expression during fruit ripening in selected genotypes identified in objectives 1 and 2, and 5. Identify and analyze regulatory mechanisms of chloroplast disassembly and chromoplast formation. During the 3 years research period we have carried out most of the research activities laid out in the original proposal and our key conclusions are as follows: 1. the engineered polyamine metabolism strategy proposed by the US collaborators can not increase lycopene content either on its own or in combination with an hp mutant (hp-2ᵈᵍ); 2. The hp-2ᵈᵍ affects strongly the transcriptional profile of the tomato fruit showing a strong tendency for up- rather than down-regulation of genes, 3. Ontology assignment of these miss-regulated genes revealed a consistent up-regulation of genes related to chloroplast biogenesis and photosynthesis in hp-2ᵈᵍ mutants throughout fruit development; 4. A tendency for up-regulation was also usually observed in structural genes involved in phytonutrientbiosynthesis; however this up-regulation was not as consistent. 5. Microscopic observations revealed a significantly higher number of chloroplasts in pericarp cells of mature-green hp-2ᵈᵍ/hp-2ᵈᵍ fruits in comparison to their normal fully isogenic counterparts. 6. The relative abundance of chloroplasts could be observed from early stages of fruit development. Cumulatively these results suggest that: 1. the overproduction of secondary metabolites, characterizing hp-2ᵈᵍ/hp-2ᵈᵍ fruits, is more due to chloroplast number rather then to transcriptional activation of structural genes of the relevant metabolic pathways, and 2. The molecular trigger increasing metabolite levels in hp-2ᵈᵍ mutant fruits should be traced at early stage of fruit development.

Begomoviruses, which constitute one genus of the Geminiviridae family, are single-stranded DNA viruses that infect many dicotyledonous crops important to large agricultural industries as well as to subsistence growers. Although all begomoviruses are transmitted by whiteflies (Bemisia tabaci), they have proven difficult to manage even with heavy insecticide applications. The begomovirus, Tomato yellow leaf curl virus (TYLCV), has been a problem in tomato production in Israel since the 1950s and in the United States since 1997. Approximately 89 begomoviruses have now been reported to infect tomato. Crop losses due to begomoviruses such as TYLCV and Tomato mottle virus (ToMoV), are limiting factors in tomato cultivation in Israel, the U.S., and many tomato-growing regions throughout the world. To overcome these limitations, we proposed a two-step strategy that combines transgenic and conventional resistance in order to develop tomato plants that are resistant to multiple begomoviruses. In the first step, we have developed transgenic tomato plants expressing trans-dominant interfering mutants Rep and C3 from TYLCV and ToMoV, and tested whether these plants are resistant to infection by these two viruses. In the second step we have tested whether pyramiding transgenic and conventional resistance is superior to either strategy alone. The specific objectives of the proposal were: 1. Design and test trans-dominant interfering constructs for TYLCV and ToMoV Rep and C3 in transient replication interference assays. 2. Generate and test transgenic tomato plants expressing mutant Rep and C3 in resistance assays. 3. Generate and test conventional resistant lines that also express mutant Rep and C3. Two viral replication interfering constructs, expressing the trans-dominant interfering mutants Rep and C3, were designed and constructed during this project. One construct, pNSB1630 was based on TYLCV sequences and the other, pNSB1682, based on ToMoV sequences. The TYLCV transformation construct was tested in a protoplasts replication assay, and was found to inhibit TYLCV replication. The ToMoV transformation construct is yet to be tested in a protoplast assay. Both transformation vectors, pNSB1630 and pNSB1682, were used to transform four different tomato lines, and generate transgenic plants. The tomato lines used for transformation were: FL7613, MM, TY172, TY199. FL7613 and MM are susceptible to both TYLCV and ToMoV. TY172 and TY199 are breeding lines developed at Volcani Center. TY172 is resistant to TYLCV but susceptible to ToMoV, while TY199 is resistant to both TYLCV and ToMoV. When transgenic T1 plants expressing the pNSB1630 constructed were screened for TYLCV resistance, it was found that these plants showed very low level of TYLCV resistance, if any. However, some of these lines showed high level of resistance to ToMoV. Only five transgenic T1 lines expressing the pNSB1682 construct were tested (so far) for resistance to ToMoV. It was found that all five lines express very high level of resistance to ToMoV. Although we haven’t finished (yet) the screen of all the transgenic lines, it is already clear that we were able to successfully combine genetic resistance for TYLCV with transgenic resistance to ToMoV.

The broad objective of the project was to develop a feasible approach to combat diarrheal disease caused by ETEC through the development of a low-cost oral immunogen in tomato fruit, expressed in the context of a prototype tomato that would answer the shortcomings of plant oral vaccines, especially in terms of produce handling and control of gene escape. Specifically, the goals for Boyce Thompson Institute (BTI) on this project were to develop transgenic tomato lines that express the enterotoxigenic E. coli (ETEC) heat-labile enterotoxin (LT) subunits A and/or B for use in oral edible vaccines, and to optimize expression and assembly of these antigens in tomato fruits.LT-B is a useful vaccine antigen against ETEC disease, since antibodies against LT-B can prevent binding and delivery of the holotoxinLT. Mutant forms of the toxic LT-A subunit that have reduced toxicity can be co-expressed and assembled with LT-Bpentamers to form mutant LT (mLT) complexes that could be used as mucosaladjuvants for other oral vaccines. Work on the project is continuing at Arizona State University, after Dr. Mason moved there in August 2002. A number of approaches were taken to ensure the expression of both subunits and bring about their assembly inside the transgenic fruits. Initially, expression was driven by the fruit-specific E-8 promoter for LT-B and the constitutive CaMV 35S promoter for LT-A(K63). While LT-B accumulated up to 7 µg per gram ripe fruit, assembled LT-K63 was only 1 µg per gram. Since promoter activities for the two genes likely differed in cell type and developmental stage specificity, the ratios of A and B subunits was not optimal for efficient assembly in all cells. In order to maximize the chance of assembly of mLT in fruit, we focused on constructs in which both genes are driven by the same promoter. These included co-expression plasmids using the 35S promoter for both, while switching to attenuated mLTs (LT-R72 and LT-G192) that have shown greater potential for oral adjuvanticity than the initial LT-K63, and thus are better candidates for a plant-derived adjuvant. Other, more novel approaches were then attempted, including several new vectors using the tomato fruit-specific E8 promoter driving expression of both LT-B and mutant LT-A, as well as a dicistronic construct for co-expression of both LT-B and mutant LT-A genes from a single promoter, and a geminivirusreplicon construct. We describe in the Appendix the results obtained in transgenic tomato lines transformed with these constructs. Overall, each contributed to enhanced expression levels, but the assembly itself of the holotoxin to high levels was not observed in the fruit tissues. The Israeli lab’s specific objective was to develop transgenic tomato lines expressing the LTholotoxin antigen bearing attributes to prevent gene escape (male sterility and orange fruit color) and to improve the dissemination of the oral vaccine (long shelf-life tomato cherry fruit or tomato processing background). Breeding lines bearing a number of attributes to prevent gene escape were developed by combining material and backcrossing either to a tomato cherry background, or two different processing backgrounds. Concomitantly, (these lines can be utilized for the creation of any future oral vaccine or other therapeutic-expressing tomato, either by crosses or transformation), the lines were crossed to the holotoxin-expressing tomatoes received from the United States, and this transgenic material was also incorporated into the backcrossing programs. To date, we have finalized the preparation of the cherry tomato material, both non-transgenic (bearing all the desired attributes), and transgenic, expressing the holotoxin. The level of expression of LT-B in the cherry fruits was comparable to the original transgenic tomatoes. Since it was not higher, this would necessitate the consumption of more fruits to reach a desired dose. A final backcross has been made for both the non-transgenic and the transgenic material in the processing lines. Auxin sprays resulted in high percentages of fruit set, but the processing genotypes gave many puffed fruits.

Clavibacter michiganensis subsp. michiganensis (Cmm), the causal agent of bacterial wilt and canker of tomato, is the most destructive bacterial disease of tomato causing substantial economic losses in Israel, the U.S.A. and worldwide. The molecular strategies that allow Cmm, a Gram-positive bacterium, to develop a successful infection in tomato plants are largely unknown. The goal of the project was to elucidate the molecular interactions between Cmmand tomato. The first objective was to analyze gene expression profiles of susceptible tomato plants infected with pathogenic and endophytic Cmmstrains. Microarray analysis identified 122 genes that were differentially expressed during early stages of infection. Cmm activated typical basal defense responses in the host including induction of defense-related genes, production of scavenging of free oxygen radicals, enhanced protein turnover and hormone synthesis. Proteomic investigation of the Cmm-tomato interaction was performed with Multi-Dimensional Protein Identification Technology (MudPIT) and mass spectroscopy. A wide range of enzymes secreted by Cmm382, including cell-wall degrading enzymes and a large group of serine proteases from different families were identified in the xylem sap of infected tomato. Based on proteomic results, the expression pattern of selected bacterial virulence genes and plant defense genes were examined by qRT-PCR. Expression of the plasmid-borne cellulase (celA), serine protease (pat-1) and serine proteases residing on the chp/tomA pathogenicity island (chpCandppaA), were significantly induced within 96 hr after inoculation. Transcription of chromosomal genes involved in cell wall degradation (i.e., pelA1, celB, xysA and xysB) was also induced in early infection stages. The second objective was to identify by VIGS technology host genes affecting Cmm multiplication and appearance of disease symptoms in plant. VIGS screening showed that out of 160 tomato genes, which could be involved in defense-related signaling, suppression of 14 genes led to increase host susceptibility. Noteworthy are the genes Snakin-2 (inhibitor of Cmm growth) and extensin-like protein (ELP) involved in cell wall fortification. To further test the significance of Snakin -2 and ELP in resistance towards Cmm, transgenic tomato plants over-expressing the two genes were generated. These plants showed partial resistance to Cmm resulting in a significant delay of the wilt symptoms and reduction in size of canker lesion compared to control. Furthermore, colonization of the transgenic plants was significantly lower. The third objective was to assess the involvement of ethylene (ET), jasmonate (JA) and salicylic acid (SA) in Cmm infection. Microarray and proteomic studies showed the induction of enzymes involved in ET and JA biosynthesis. Cmm promoted ET production 8 days after inoculation and SIACO, a key enzyme of ET biosynthesis, was upregulated. Inoculation of the tomato mutants Never ripe (Nr) impaired in ET perception and transgenic plants with reduced ET synthesis significantly delayed wilt symptoms as compared to the wild-type plants. The retarded wilting in Nr plants was shown to be a specific effect of ET insensitivity and was not due to altered expression of defense related genes, reduced bacterial population or decrease in ethylene biosynthesis . In contrast, infection of various tomato mutants impaired in JA biosynthesis (e.g., def1, acx1) and JA insensitive mutant (jai1) yielded unequivocal results. The fourth objective was to determine the role of cell wall degrading enzymes produced by Cmm in xylem colonization and symptoms development. A significance increase (2 to 7 fold) in expression of cellulases (CelA, CelB), pectate lyases (PelA1, PelA2), polygalacturonase and xylanases (XylA, XylB) was detected by qRT-PCR and by proteomic analysis of the xylem sap. However, with the exception of CelA, whose inactivation led to reduced wilt symptoms, inactivation of any of the other cell wall degrading enzymes did not lead to reduced virulence. Results achieved emphasized the complexity involved in Cmm-tomato interactions. Nevertheless they provide the basis for additional research which will unravel the mechanism of Cmm pathogenicity and formulating disease control measures.

This proposal is focused on examination of two plant interactions: parasitic with Orobanche, and symbiosis with arbuscular mycorrhiza fungi (AMF), and the involvement of a newly define plant hormones, strigolactones (SLs), in these plant interactions. In addition to strigolactones role in regulation of above-ground plant architecture, they are also known to be secreted from roots, and to be a signal for seed germination of the parasitic plants Orobanche. Moreover, secreted strigolactones were recognized as inducers of AMFhyphae branching. The present work was aimed at Generation of RNAi mutants of both tomato and Medicago, targeting multiple genes that may be involved in strigolactone production, carotenoid biosynthesis pathway, Pi signaling or other metabolic pathways, and hence affect AMF colonization and/or Orobanche resistance. Following the newly formed and existing RNAi mutants were examined for AMF colonization and Orobanche resistance. At the first phase of this project Orobanche seed germination assays and AMF colonization were examined in intact plants. These assays were shown to be effective and resulted with enhancement of Orobanche seed germination and AMF colonization in WT tomato plants, whereas roots of strigolactones impaired lines did not result with Orobanche seed germination and mycorrhiza colonization. Unexpectedly, root organ cultures (ROC) that were produced from the same wild type (WT) and mutant lines did not induce the Orobanche seed germination and AMFhyphal branching. This implies that under in vitro conditions ROC cultures are missing an important component for induction of Orobanche seed germination and AMFhyphal branching. In another line of experiments we have tested transgenic lines of Medicagotruncatula for AMFhuyphal branching and Orobanche seed germination assays. These lines included lines silenced for a GRAS transcription factor (RNAi 1845), an NBS-LRR type resistance gene (RNAi 1847), a kinase (RNAi 2403) and a protein of unknown function (RNAi 2417). In all cases, five independent transgenic root lines showed altered AMFphenotypes with reduced or aberrant colonization patterns. Following, we transformed tomato plants with the M. truncatulaTC 127050 PhosphoinositidekinaseRNAi construct. Transgenic lines that contained GUS constructs were used as control. All transgenic lines showed reduced level of Orobanche seed germination, masking any strigoalctones-specific effect. The research demonstrated that SLs production may not be examined in ROC –based bioassays. It was shown by the 3 independent assays employed in this project that none of the recognized characters of SLs may be reflected in these bioassays. However, when the whole plant root exudates were examined, SLs activity in root exudates was demonstrated. Hence, it can be concluded that the presence of an intact shoot, and possibly, shoot factors, may be necessary for production of SLs in roots. Another point of interest that rises from these results is that the presence of SLs is not necessary for AMF completion of life cycle. Hence, it may be concluded that SLs are important for AMFhyphal branching, before symbiosis, but not essential for AMF colonization and life cycle completion under ROC system conditions.

Fungal plant pathogens are responsible for extensive annual crop and revenue losses throughout the world. To better understand why fungi cause diseases, we performed gene-disruption mutagenesis on several pathogenic Colletotrichum species and demonstrated that pathogenic isolates can be converted to symbionts expressing non-pathogenic lifestyles. One group of nonpathogenic mutants confer disease protection against pathogenic species of Col!etotrichum, Fusarium and Phytophthora; drought tolerance; and growth enhancement to host plants. These mutants have been defined as mutualists and disease resistance correlates to a decrease in the time required for hosts to activate defense systems when exposed to virulent fungi. A second group of non-pathogenic mutants did not confer disease resistance and were classified as commensals. In addition, we have demonstrated that wildtype pathogenic Colletotrichum species can express non-pathogenic lifestyles, including mutualism, on plants they colonize asymptomatically. We have been using wildtype and isogenic gene disruption mutants to characterize gene expression patterns in plants colonized with a pathogen, mutualist or commensal. The US group is contrasting genes expressed during colonization by mutuahstic and commensal mutants of C. magna and a pathogenic wildtype C. coccodes on tomato. The Israeli group is characterizing genes expressed during asymptomatic colonization of tomato by wildtype C. acutatum and a non-pathogenic mutant.To accomplish this we have been utilizing suppressive subtraction hybridization, microarray and sequencing strategies. The expected contribution of this research to agriculture in the US and Israel is: 1) understanding how pathogens colonize certain hosts asymptomatic ally will shed light on the ecology of plant pathogens which has been described as a fundamental deficiency in plant pathology; 2) identifying genes involved in symbiotically conferred disease resistance will help explain why and how pathogens cause disease, and may identify new candidate targets for developing genetically modified disease resistant crop plants.