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Several strategies are required to achieve satisfactory control of Asian soybean rust (ASR) caused by the fungus Phakopsora pachyrhizi. The use of resistance inducers represents a helpful alternati...

The virulence factors (VF) that parasitoids use to regulate the growth and development of their hosts have potential as new events in transgenic plants. VFs are promising candidates for biotechnological exploitation and implementation of pest-control tools to meet the world’s demand for food in 2100, while sustainably increasing agricultural production. However, VFs have not been used in commercial transgenic plants, since they are thought to target only a narrow range of insects, due to the parasitoid host specialization. We investigated the potential of chitolectin (TnChit), produced by the teratocytes of Toxoneuron nigriceps (Viereck) (Hymenoptera: Braconidae), as a transgenic event to control several non-permissive hosts: Chrysodeixis includens (Walker), Spodoptera albula (Walker), Spodoptera frugiperda (JE Smith) (Lepidoptera: Noctuidae), Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) and Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae). Third instars of C. includens, S. albula and S. frugiperda had lower survival and probability of molting on TnChit-modified plants than on control plants. The nutritional indices of S. frugiperda were also altered. Larval survival and pupal weight of males of T. absoluta were reduced on transgenic plants. Adult emergence of B. tabaci was also reduced. The expression of the TnChit gene differed in the transformation events obtained and was consistent with the biological effects observed. The effects of TnChit on non-permissive hosts demonstrate the potential of the virulence factors that parasitoids employ in host regulation, as a source of new genes in plant transformation for control of insect pests.

The transition from flowering to fruit production, namely fruit set, is crucial to ensure successful sexual plant reproduction. Although studies have described the importance of hormones (i.e. auxin and gibberellins) in controlling fruit set after pollination and fertilization, the role of microRNA-based regulation during ovary development and fruit set is still poorly understood. Here we show that the microRNA159/GAMYB1 and -2 pathway (the miR159/GAMYB1/2 module) is crucial for tomato ovule development and fruit set. MiR159 and SlGAMYBs were expressed in preanthesis ovaries, mainly in meristematic tissues, including developing ovules. SlMIR159-overexpressing tomato cv. Micro-Tom plants exhibited precocious fruit initiation and obligatory parthenocarpy, without modifying fruit shape. Histological analysis showed abnormal ovule development in such plants, which led to the formation of seedless fruits. SlGAMYB1/2 silencing in SlMIR159-overexpressing plants resulted in misregulation of pathways associated with ovule and female gametophyte development and auxin signalling, including AINTEGUMENTA-like genes and the miR167/SlARF8a module. Similarly to SlMIR159-overexpressing plants, SlGAMYB1 was downregulated in ovaries of parthenocarpic mutants with altered responses to gibberellins and auxin. SlGAMYBs likely contribute to fruit initiation by modulating auxin and gibberellin responses, rather than their levels, during ovule and ovary development. Altogether, our results unveil a novel function for the miR159-targeted SlGAMYBs in regulating an agronomically important trait, namely fruit set.

Auxin modulates a range of plant developmental processes including embryogenesis, organogenesis, and shoot and root development. Recent studies have shown that plant hormones also strongly influence metabolic networks, which results in altered growth phenotypes. Modulating auxin signalling pathways may therefore provide an opportunity to alter crop performance. Here, we performed a detailed physiological and metabolic characterization of tomato (Solanum lycopersicum) mutants with either increased (entire) or reduced (diageotropica-dgt) auxin signalling to investigate the consequences of altered auxin signalling on photosynthesis, water use, and primary metabolism. We show that reduced auxin sensitivity in dgt led to anatomical and physiological modifications, including altered stomatal distribution along the leaf blade and reduced stomatal conductance, resulting in clear reductions in both photosynthesis and water loss in detached leaves. By contrast, plants with higher auxin sensitivity (entire) increased the photosynthetic capacity, as deduced by higher V

Made available in DSpace on 2018-12-11T17:37:26Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-09-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Key message: A seed maturation protein gene (CaSMP) from Coffea arabica is expressed in the endosperm of yellow/green fruits. The CaSMP promoter drives reporter expression in the seeds of immature tomato fruits. Abstract: In this report, an expressed sequence tag-based approach was used to identify a seed-specific candidate gene for promoter isolation in Coffea arabica. The tissue-specific expression of the cognate gene (CaSMP), which encodes a yet uncharacterized coffee seed maturation protein, was validated by RT-qPCR. Additional expression analysis during coffee fruit development revealed higher levels of CaSMP transcript accumulation in the yellow/green phenological stage. Moreover, CaSMP was preferentially expressed in the endosperm and was down-regulated during water imbibition of the seeds. The presence of regulatory cis-elements known to be involved in seed- and endosperm-specific expression was observed in the CaSMP 5′-upstream region amplified by genome walking (GW). Additional histochemical analysis of transgenic tomato (cv. Micro-Tom) lines harboring the GW-amplified fragment (~ 1.4 kb) fused to uidA reporter gene confirmed promoter activity in the ovule of immature tomato fruits, while no activity was observed in the seeds of ripening fruits and in the other organs/tissues examined. These results indicate that the CaSMP promoter can be used to drive transgene expression in coffee beans and tomato seeds, thus representing a promising biotechnological tool. Department of Genetics Institute of Biosciences Sao Paulo State University (UNESP) Department of Biological Sciences (LCB) Escola Superior de Agricultura “Luiz de Queiroz” (ESALQ) University of Sao Paulo (USP) Embrapa Coffee and Coffee Center Alcides Carvalho Agronomic Institute of Campinas Department of Genetics Institute of Biosciences Sao Paulo State University (UNESP) FAPESP: 2009/54361-9

Uma das abordagens mais utilizadas atualmente para se estudar o metabolismo e a transdução de sinais hormonais é o uso de mutantes com alterações nos genes que codificam os principais componentes desses processos. A cultivar miniatura de tomateiro (Lycopersicon esculentum) denominada Micro-Tom (MT) possui porte reduzido (8 cm) e ciclo de apenas 75 dias, constituindo-se em um excelente modelo para uma abordagem genética de estudos fisiológicos. Mutantes com alterações no balanço auxina/citocinina, ou na capacidade de resposta a esse balanço, podem ser utilizados para desvendar o papel da interação entre esses hormônios no controle do desenvolvimento, inclusive no que se refere à capacidade de regeneração in vitro. O presente trabalho teve como objetivo criar um modelo para se estudar o papel do balanço auxina/citocinina endógeno na competência para regeneração in vitro, através da incorporação das mutações dgt, brt, gf, lutescent, ls e bu, as quais sugerem alterações no metabolismo e/ou sensibilidade hormonal, na cultivar MT. Essas mutações foram caracterizadas quanto à sensibilidade à auxina e citocinina através da obtenção de curvas de dose-resposta, utilizando-se diferentes concentrações de AIA (0; 0.1; 1; 10 e 100 µM) e TDZ (0; 0.01; 0.1; 1; 10 e 100 µM), em segmentos de pecíolo e plântulas germinadas em gerbox, respectivamente. As mutações caracterizadas para auxina e citocinina, bem como o controle MT, foram testadas in vitro, quanto à sua capacidade de regeneração, em meio de cultura MS, suplementado com 5µM de BAP (explantes cotiledonares e segmentos de hipocótilo) e 4,5 µM de Zeatina (segmentos de raiz). Os mutantes lutescent, gf e bu, apesar de possuírem fenótipo bastante interessante, não parecem ser relacionados à sensibilidade à auxina e citocinina, bem como ao etileno. Já os mutantes dgt e brt apresentaram respostas típicas de mutantes com pouca sensibilidade à auxina e citocinina, respectivamente. Dessa forma, a resposta ao balanço AIA/Cks é alterado nesses mutantes, afetando o processo de regeneração. Nos segmentos de hipocótilo, houve formação de calos em todos os explantes, entretanto, poucos explantes formaram gemas adventícias, sendo que o mutante dgt foi o que apresentou menor taxa de regeneração. Nos explantes cotiledonares, a taxa de regeneração foi menor ainda no MT e no mutante dgt, e ausente no mutante brt. Não houve regeneração em nenhum dos genótipos em segmentos de raiz. Esses resultados sugerem que, embora a competência para regeneração in vitro possa ser dependente do balanço AIA/Cks, ou da resposta a esse balanço, existem interações mais complexas entre esses hormônios e seus efeitos na regeneração. A exemplo disso, era de se esperar uma maior formação de gemas caulinares em dgt, já que sua capacidade de resposta ao balanço está voltada para Cks. Um estudo mais aprofundado para a correta interpretação dessa interação deve levar em conta que a sensibilidade à auxina é necessária também no processo de desdiferenciação. Além disso, auxinas e citocininas não só atuam na diferenciação, mas também podem interferir na regeneração, independente do balanço AIA/Cks, já que são necessárias para a expansão e divisão celular. To date, one of approaches more used to study the metabolism and transduction of hormone signals are the mutants with alterations in genes which encode the principal components of this process. The miniature cultivar of tomato (Lycopersicon esculentum) named Micro-Tom (MT) possesses reduced size (8 cm) and a life cycle of just 75 days, constituting an excellent model for a genetic approach of physiology studies. Mutants with altered auxin/cytokinin ratio, or in the response capacity to this ratio, can be used to clear up the role of interactions between these hormones in the control of development, including those concerning the in vitro regeneration capacity. The current work aimed to produce a model to study the role of endogenous auxin/cytokinins ratio in the competence for in vitro regeneration, through incorporation of the dgt, brt, gf, lutescent, ls and bu mutations, which concern altered hormone metabolism and/or sensibility, in the MT cultivar. These mutations were tested for the hypothesis of being related to auxin and cytokinin sensibility by means of dose-response curves, using different concentrations of IAA (0; 0.1; 1; 10 e 100 µM) and TDZ (0; 0.01; 0.1; 1; 10 e 100 µM), in petiole segments and gerbox-germinated seedlings, respectively. The mutations confirmed to be auxin and cytokinin related, as well the MT control, were tested in vitro for regeneration capacity, in MS culture media, with either 5µM de BAP (cotyledon explants and hypocotyl segments) or 4,5 µM Zeatin (root segments). The luescent, gf and bu mutants, despite showing an interesting phenotype, do not seem to be related to auxin and cytokinin sensibility, as well as to ethylene. However, the dgt and brt mutants showed typical responses of reduced auxin and cytokinin sensibility, respectively. Thus, the response to auxin/cytokinin is altered in these mutants, affecting the regeneration process. Calli were observed in all hypocotyl segments, however few explants formed adventitious buds, with the dgt mutant presenting the least regeneration ratio. In the cotyledon explants, the regeneration ratio was even less than in MT and in dgt, and absent in the brt mutant. There was no regeneration whatsoever from root segments in any genotype. These results suggest that, although the competence for in vitro regeneration might be dependent on the IAA/Cks ratio, or the response to this ratio, interactions far more complex exist between these hormones and their effects on regeneration. For instance, a greater adventitious buds formation was expected in dgt, because its response ability is biased toward Cks. A deeper study for the correct interpretation of these data should consider that the sensibility to auxin is also necessary in the process of undifferentiation. Besides, both auxins and cytokinins have roles not only in differentiation, but also can interfere with regeneration independent of the IAA/Cks ratio, as they are required for cell division and expansion.

Genetic transformation using mature tissue as explant is important for the precocious evaluation of the genetically modified characteristic. This research presents a protocol for adventitious organogenesis and genetic transformation of sweet orange varieties ( Citrus sinensis L. Osbeck) using as explants internodal segments from adult plants maintained in the greenhouse. Organogenesis was induced in DBA3 medium supplemented with benzilaminopurine (1.0 mg l −1 )+naphthaleneacetic acid (0.5 mg l −1 ), in the dark at 27±2 °C until adventitious buds had formed and then transferred to a 16-h photoperiod. Histological analysis showed an indirect formation of the adventitious buds from callus that had proliferated at the cut ends of the explants. Transformation was done using the Agrobacterium tumefaciens strain EHA-105 containing the plasmid p35SGUSINT as a vector. The explants were co-cultivated with Agrobacterium for 24 h, at 24 °C. Adventitious shoots were tested histochemically for GUS expression and micrografted into plantlets of the rootstock Carrizo citrange ( C. sinensis L. Osbeck× Poncirus trifoliata L. Raf.). Genetic transformation was confirmed by PCR and Southern blot analysis of the recovered plants.

Made available in DSpace on 2013-08-12T18:46:36Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-09-01 Made available in DSpace on 2013-09-30T18:37:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-09-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:50:24Z No. of bitstreams: 0 Made available in DSpace on 2014-05-20T13:50:24Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-09-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Despite the wide use of plant regeneration for biotechnological purposes, the signals that allow cells to become competent to assume different fates remain largely unknown. Here, it is demonstrated that the Regeneration1 (Rg1) allele, a natural genetic variation from the tomato wild relative Solanum peruvianum, increases the capacity to form both roots and shoots in vitro; and that the gibberellin constitutive mutant procera (pro) presented the opposite phenotype, reducing organogenesis on either root-inducing medium (RIM) or shoot-inducing medium (SIM). Mutants showing alterations in the formation of specific organs in vitro were the auxin low-sensitivity diageotropica (dgt), the lateral suppresser (ls), and the KNOX-overexpressing Mouse ears (Me). dgt failed to form roots on RIM, Me increased shoot formation on SIM, and the high capacity for in vitro shoot formation of ls contrasted with its recalcitrance to form axillary meristems. Interestingly, Rg1 rescued the in vitro organ formation capacity in proRg1 and dgtRg1 double mutants and the ex vitro low lateral shoot formation in pro and ls. Such epistatic interactions were also confirmed in gene expression and histological analyses conducted in the single and double mutants. Although Me phenocopied the high shoot formation of Rg1 on SIM, it failed to increase rooting on RIM and to rescue the non-branching phenotype of ls. Taken together, these results suggest REGENERATION1 and the DELLA mutant PROCERA as controlling a common competence to assume distinct cell fates, rather than the specific induction of adventitious roots or shoots, which is controlled by DIAGEOTROPICA and MOUSE EARS, respectively. Univ São Paulo, ESALQ, Dept Biol Sci LCB, Lab Hormonal Control Plant Dev, BR-13418900 Piracicaba, SP, Brazil Univ São Paulo, Lab Plant Breeding, CENA, BR-13400970 Piracicaba, SP, Brazil Univ Estadual Paulista, Inst Biol, Dept Genet, Lab Mol Genet Plant Dev, BR-18618970 Botucatu, SP, Brazil Univ Estadual Paulista, Inst Biol, Dept Genet, Lab Mol Genet Plant Dev, BR-18618970 Botucatu, SP, Brazil FAPESP: 07/07175-0

Background The tomato (Solanum lycopersicum L.) plant is both an economically important food crop and an ideal dicot model to investigate various physiological phenomena not possible in Arabidopsis thaliana. Due to the great diversity of tomato cultivars used by the research community, it is often difficult to reliably compare phenotypes. The lack of tomato developmental mutants in a single genetic background prevents the stacking of mutations to facilitate analysis of double and multiple mutants, often required for elucidating developmental pathways. Results We took advantage of the small size and rapid life cycle of the tomato cultivar Micro-Tom (MT) to create near-isogenic lines (NILs) by introgressing a suite of hormonal and photomorphogenetic mutations (altered sensitivity or endogenous levels of auxin, ethylene, abscisic acid, gibberellin, brassinosteroid, and light response) into this genetic background. To demonstrate the usefulness of this collection, we compared developmental traits between the produced NILs. All expected mutant phenotypes were expressed in the NILs. We also created NILs harboring the wild type alleles for dwarf, self-pruning and uniform fruit, which are mutations characteristic of MT. This amplified both the applications of the mutant collection presented here and of MT as a genetic model system. Conclusions The community resource presented here is a useful toolkit for plant research, particularly for future studies in plant development, which will require the simultaneous observation of the effect of various hormones, signaling pathways and crosstalk.

Os hormônios auxina e citocininas são essenciais ao desenvolvimento das plantas, pois controlam os processos de divisão, expansão e diferenciação celular, os quais, por sua vez, influenciam desde a formação do embrião até o amadurecimento dos frutos e senescência. Auxinas e citocininas regulam respostas fisiológicas comuns, sugerindo haver múltiplos mecanismos de interação. Neste trabalho, um modelo para estudar a interação entre auxinas e citocininas no controle do desenvolvimento é proposto, sendo baseado em plantas mutantes e transgênicas, além de duplos mutantes, com alterações na sensibilidade ou metabolismo de auxina e citocinina. O mutante bushy root (brt) foi caracterizado como pouco sensível à citocinina e sugere uma importante função da citocinina no desenvolvimento da semente e na determinação da dominância apical em tomateiro. Os mutantes potato leaf (c) e entire (e) foram introgredidos no background Micro-Tom (MT), caracterizados e utilizados para verificar como eles afetam a sensibilidade à auxina e como interferem no desenvolvimento da planta. Esses mutantes foram comparados com MT, com o mutante diageotropica (dgt), que é pouco sensível à auxina, e com os duplos mutantes c dgt e dgt e. A mutação c não alterou a sensibilidade à auxina e estaria influenciando apenas a arquitetura foliar. O mutante e parece ser mais sensível à auxina com relação à capacidade de formar raízes in vitro e partenocarpia. O duplo mutante dgt e mostrou fenótipo aditivo (intermediário), sugerindo que DGT e E agem em vias paralelas na resposta à auxina. Foram criadas linhagens transgênicas com superexpressão da enzima citocinina oxidase de Arabidopsis (35S:AtCKX2), que resulta em plantas com baixos níveis endógenos de citocinina. As linhagens transgênicas (MT CKX2 e dgt CKX2) foram comparadas com MT, brt, dgt e brt dgt. A maioria dos parâmetros que estavam relacionados com o desenvolvimento vegetativo, e todos os parâmetros relacionados ao desenvolvimento reprodutivo, foram associados ao efeito dos níveis absolutos de auxina e citocinina, sendo provavelmente resultantes de alterações nos processos de divisão e expansão celular. A dominância apical e a morfogênese in vitro (formação de gemas caulinares e raízes) foram associadas ao efeito do balanço auxina/citocinina, sendo esses processos conhecidamente regulados pelo efeito dessas classes hormonais na diferenciação celular The plant hormones auxin and cytokinin are crucial for plant development, since they control cell division, expansion and differentiation, which regulate developmental processes starting from embryo formation to fruit ripening and senescence. Auxins and cytokinins usually regulate the same physiological responses, suggesting multiple mechanisms of interaction between these hormones. In this work, a model to study auxin and cytokinin interaction in the control of plant development is proposed, based on mutants, transgenic lines and double mutants with alterations on the auxin and cytokinin sensitivity or metabolism. The bushy root (brt) mutant was characterized as having low cytokinin sensitivity and suggested a relevant function to cytokinin in the seed development and apical dominance in tomato. The potato leaf (c) and entire (e) mutants were introgressed in the Micro-Tom background, characterized and used to check how they affect the auxin sensitivity and the plant development. The responses from these mutants were compared to MT and diageotropica (dgt), which is a low sensitive auxin mutant, and also compared to c dgt and dgt e double mutants. The c mutation did not alter the auxin sensitivity and was only related to leaf architecture. The e mutant seemed to be more sensitive to auxin in the in vitro root induction and parthenocarpy. The double mutant dgt e showed an additive phenotype, suggesting that DGT and E act in parallel pathways controlling auxin sensitivity. Transgenic lines were generated overexpressing the cytokinin oxidase from Arabidopsis (35S:AtCKX2), which renders plants with low endogenous cytokinin levels. Transgenic lines (MT CKX2 and dgt CKX2) were compared to MT, brt, dgt and brt dgt. Most of the traits related to vegetative development and all traits related to reproductive development were linked to the effect of auxin and cytokinin absolute levels, probably reflecting to the effect of these hormones in cell division and expansion. Apical dominance and in vitro morphogenesis (shoot and root formation) were linked to the auxin-to-cytokinin ratio, since these processes are well known to be regulated by the effect of these two hormones in cell differentiation

In this report, an expressed sequence tag-based approach was used to identify a seed-specific candidate gene for promoter isolation in Coffea arabica. The tissue-specific expression of the cognate gene (CaSMP), which encodes a yet uncharacterized coffee seed maturation protein, was validated by RT-qPCR. Additional expression analysis during coffee fruit development revealed higher levels of CaSMP transcript accumulation in the yellow/green phenological stage. Moreover, CaSMP was preferentially expressed in the endosperm and was down-regulated during water imbibition of the seeds. The presence of regulatory cis-elements known to be involved in seed- and endosperm-specific expression was observed in the CaSMP 5?-upstream region amplified by genome walking (GW). Additional histochemical analysis of transgenic tomato (cv. Micro-Tom) lines harboring the GW-amplified fragment (~ 1.4 kb) fused to uidA reporter gene confirmed promoter activity in the ovule of immature tomato fruits, while no activity was observed in the seeds of ripening fruits and in the other organs/tissues examined. These results indicate that the CaSMP promoter can be used to drive transgene expression in coffee beans and tomato seeds, thus representing a promising biotechnological tool. Made available in DSpace on 2019-10-21T18:04:28Z (GMT). No. of bitstreams: 1 Identificactionofaseedmaturationprotein.pdf: 1721993 bytes, checksum: aef8a71920ca8c5b2d24829ef318ce59 (MD5) Previous issue date: 2018