Molecular signals required for type III secretion and translocation of the Xanthomonas campestris AvrBs2 protein to pepper plants

Strains of Xanthomonas campestris pv. vesicatoria (Xcv) carrying avrBs2 are specifically recognized by Bs2 pepper plants, resulting in localized cell death and plant resistance. Agrobacterium-mediated transient expression of the Xcv avrBs2 gene in plant cells results in Bs2-dependent cell death, indicating that the AvrBs2 protein alone is sufficient for the activation of disease resistance-mediated cell death in planta. We now provide evidence that AvrBs2 is secreted from Xcv and that secretion is type III (hrp) dependent. N- and C-terminal deletion analysis of AvrBs2 has identified the effector domain of AvrBs2 recognized by Bs2 pepper plants. By using a truncated Pseudomonas syringae AvrRpt2 effector reporter devoid of type III signal sequences, we have localized the minimal region of AvrBs2 required for type III secretion in Xcv. Furthermore, we have identified the region of AvrBs2 required for both type III secretion and translocation to host plants. The mapping of AvrBs2 sequences sufficient for type III delivery also revealed the presence of a potential mRNA secretion signal.

Cell-Specific Production and Antimicrobial Activity of Naphthoquinones in Roots of Lithospermum erythrorhizon1

Pigmented naphthoquinone derivatives of shikonin are produced at specific times and in specific cells of Lithospermum erythrorhizon roots. Normal pigment development is limited to root hairs and root border cells in hairy roots grown on “noninducing” medium, whereas induction of additional pigment production by abiotic (CuSO4) or biotic (fungal elicitor) factors increases the amount of total pigment, changes the ratios of derivatives produced, and initiates production of pigment de novo in epidermal cells. When the biological activity of these compounds was tested against soil-borne bacteria and fungi, a wide range of sensitivity was recorded. Acetyl-shikonin and β-hydroxyisovaleryl-shikonin, the two most abundant derivatives in both Agrobacterium rhizogenes-transformed “hairy-root” cultures and greenhouse-grown plant roots, were the most biologically active of the seven compounds tested. Hyphae of the pathogenic fungi Rhizoctonia solani, Pythium aphanidermatum, and Nectria hematococca induced localized pigment production upon contact with the roots. Challenge by R. solani crude elicitor increased shikonin derivative production 30-fold. We have studied the regulation of this suite of related, differentially produced, differentially active compounds to understand their role(s) in plant defense at the cellular level in the rhizosphere.

Stable transfer of intact high molecular weight DNA into plant chromosomes.

In conjunction with an enhanced system for Agrobacterium-mediated plant transformation, a new binary bacterial artificial chromosome (BIBAC) vector has been developed that is capable of transferring at least 150 kb of foreign DNA into a plant nuclear genome. The transferred DNA appears to be intact in the majority of transformed tobacco plants analyzed and is faithfully inherited in the progeny. The ability to introduce high molecular weight DNA into plant chromosomes should accelerate gene identification and genetic engineering of plants and may lead to new approaches in studies of genome organization.

Two different but related mechanisms are used in plants for the repair of genomic double-strand breaks by homologous recombination.

Genomic double-strand breaks (DSBs) are key intermediates in recombination reactions of living organisms. We studied the repair of genomic DSBs by homologous sequences in plants. Tobacco plants containing a site for the highly specific restriction enzyme I-Sce I were cotransformed with Agrobacterium strains carrying sequences homologous to the transgene locus and, separately, containing the gene coding for the enzyme. We show that the induction of a DSB can increase the frequency of homologous recombination at a specific locus by up to two orders of magnitude. Analysis of the recombination products demonstrates that a DSB can be repaired via homologous recombination by at least two different but related pathways. In the major pathway, homologies on both sides of the DSB are used, analogous to the conservative DSB repair model originally proposed for meiotic recombination in yeast. Homologous recombination of the minor pathway is restricted to one side of the DSB as described by the nonconservative one-sided invasion model. The sequence of the recombination partners was absolutely conserved in two cases, whereas in a third case, a deletion of 14 bp had occurred, probably due to DNA polymerase slippage during the copy process. The induction of DSB breaks to enhance homologous recombination can be applied for a variety of approaches of plant genome manipulation.

Arabidopsis mutants deficient in T-DNA integration.

Arabidopsis thaliana mutants originally isolated as hypersensitive to irradiation were screened for the ability to be transformed by Agrobacterium transferred DNA (T-DNA). One of four UV-hypersensitive mutants and one of two gamma-hypersensitive mutants tested showed a significant reduction in the frequency of stable transformants compared with radioresistant controls. In a transient assay for T-DNA transfer independent of genomic integration, both mutant lines took up and expressed T-DNA as efficiently as parental lines. These lines are therefore deficient specifically in stable T-DNA integration and thus provide direct evidence for the role of a plant function in that process. As radiation hypersensitivity suggests a deficiency in repair of DNA damage, that plant function may be one that is also involved in DNA repair, possibly, from other evidence, in repair of double-strand DNA breaks.

The potential application of pyroligneous acid in the UK agricultural industry

This paper highlights the potential benefits of smoke recovery from the production of biochar in crop management through it application as an antimicrobial agent against plant disease and as a pesticide. The study reports on the findings of zone inhibition assays on selected plant pathogens (Agrobacterium tumefacien and Xanthomonas campestris), growth studies on selected plants (Raphanus sativus and Vicia faba), and toxicity studies performed on arthropods (Myzus persicae and Tetranychus urticae). The results suggested a strong benefit to crop management in terms of crop protection against selected causal agents responsible for plant disease, with zones of inhibition observed on both Agrobacterium tumefacien and Xanthomonas campestris when inoculated with pyroligneous acid (liquid smoke) at 10% dilution. Similarly, its potential as a bio-pesticide are favorable, with a reported 20%–30% of arthropods affected (knocked out/mortality) after exposure for 48 hours.

Pyroligneous acid:a farmer’s flexible friend?

This article is a review of the findings of key studies into the potential benefits of pyroligneous acid, arising from charcoal production, to the agricultural industry. Through a review of bioassay studies conducted on known plant and human pathogens (e.g., Agrobacterium tumefacien and Xanthomonas campestris) and arthropods, and germination studies on selected crops, the article highlights a number of potential benefits of smoke recovery in the production of charcoal. In addition, the article calls for further research into the impact, if any, of its long-term use on environmental receptors/humans and for the development of a methodology to guarantee consistency in product composition, quality, and efficacy. In doing so, it is hoped that its widespread use as part of sustainable management practices adopted in the agricultural industry will be secured.

Adventitious rooting of conifers: influence of biological factors

Vegetative propagation of superior conifer trees can be achieved e.g. through rooted cuttings or rooted microshoots, the latter predominantly through in vitro tissue culture. Both techniques are used to achieve rapid multiplication of trees with favorable genetic combinations and to capture a large proportion of the genetic diversity in a single generation cycle. However, adventitious rooting of shoots (cuttings) is often not efficient due to various problems such as scarcity of roots and cessation of their growth, both of which limit the application of vegetative propagation in some conifer species. Many factors are involved in the adventitious rooting of shoots including physical and chemical ones such as plant growth regulators, carbohydrates, light quality, temperature and rooting substrates or media (reviewed by Ragonezi et al. 2010). The focus of this review is on biological factors, such as inoculations with Agrobacterium rhizogenes, plant- growth-promoting rhizobacteria and other endophytes, and mycorrhizal fungi, which were found to stimulate adventitious rooting. These microorganisms could contribute not only to adventitious root development but also help in protecting conifer plants against pathogenic microorganisms, facilitate acclimation and transplanting, and contribute to more sustainable, chemical-free forests.

Efficient genetic transformation and regeneration system from hairy root of Origanum vulgare.

2016

Superexpressão em tabaco do gene Pstol1 de arroz e de seus homólogos em milho e sorgo.

2016

Study of Rapid Alkalinization Factor (RALF)genes as plant susceptibility agents during plant-pathogen interaction in strawberry

Rapid Alkalinization Factor (RALF) are cysteins-rich peptides ubiquitous in plant kingdom. They play multiple roles as hormone signals and recently their involvement in host-pathogen crosstalk as negative regulator of immunity in Arabidopsis has also been recognized. In addition, RALF homologue peptides are secreted by different fungal pathogens as effectors during early stages of infections. The aim of this work was to characterize RALF genes as susceptibility factors during plant pathogen interaction in strawberry. For this, the genomic organization of the RALF gene families in the octoploid strawberry (Fragaria × ananassa) and the re-annotated genome of Fragaria vesca were described , identifying 13 member in F. vesca (FvRALF) and 50 members in F. x ananassa (FaRALF). The changes in expression of fruit FaRALF genes was investigated upon infection with C.acutatum and B. cinerea showing that, among RALF genes expressed in fruit, FaRALF3 was the only one upregulated by fungal infection in the ripe stage. A role of FaRALF3 as susceptibility gene was then assessed trough Agrobacterium-mediated transient FaRALF3 overexpression and silencing in fruits, revealing that FaRALF3 expression promotes fungal growth and hyphae penetration in host tissues. In silico analysis was used to identify distinct pathogen inducible elements upstream of the FaRALF3 gene. Agroinfiltration of strawberry fruit with deletion constructs of the FaRALF3 promoter identified a 5’ region required for FaRALF3 expression in fruit, but failed to identify a region responsible for fungal induced expression. Furthermore, FaRALF3 and strawberry receptor FERONIA (FaMRLK47) were heterologously expressed in E. coli in order to purify active proteins forms and study RALF-FERONIA interaction in strawberry. However, it was not possible to obtain pure and active proteins. Finally RNAi transgenic plants silenced for the FvRALF13 gene were genotypically and phenotypically characterized suggesting a role of FvRALF13 in flowering time regulation and reproductive organs development.

Genetic and process engineering approaches for improving lipid productivity in the non-conventional oleaginous yeast Cutaneotrichosporon oleaginosus

Gli oli microbici stanno ricevendo sempre più attenzioni come possibile alternativa agli oli vegetali, nel processo di sostituzione dei combustibili fossili. Tuttavia, diversi aspetti necessitano di essere ottimizzati al fine di ottenere oli economicamente competitivi e con caratteristiche chimico-fisiche desiderate. In questa ricerca, sono stati utilizzati due differenti approcci per poter realizzare l’obiettivo preposto. Il primo, si è basato sull’ingegnerizzazione genetica del lievito C. oleaginous, al fine di incrementare la produttività di lipidi e modificare la composizione dei trigliceridi (TAG) sintetizzati. Un protocollo basato su una trasformazione genetica mediata da Agrobacterium è stato utilizzato per sovraesprimere la diacilglicerol trasnferasi (DGA1), l’enzima responsabile dell’ultimo step della sintesi dei TAG, e la Δ9-desaturasi, l’enzima che catalizza la conversione dell’acido stearico (C18:0) in acido oleico (C18:1). La selezione di colonie positive e l’analisi dei mutanti ottenuti ha confermato la buona riuscita della trasformazione. Il secondo approccio ha mirato a studiare l’influenza sulla crescita e sul profilo di lipidi accumulati da C. oleaginous da parte di diversi acidi grassi volatili (VFAs), una materia prima ottenibile da trattamenti di scarti industriali. A questo proposito, sono state utilizzate fermentazioni fed-batch su scala da 1-L basate su glucosio e miscele sintetiche di acido acetico e di VFAs come fonte di carbonio. L’utilizzo simultaneo di acido acetico e acidi secondari ha mostrato come sia possibile stimolare il metabolismo microbico al fine di incrementare l'accumulo di oli e ottenere una composizione chimica lipidica desiderata.