Inés Ponce de León

Erwinia carotovora elicitors and Botrytis cinerea activate defense responses in Physcomitrella patens

BackgroundVascular plants respond to pathogens by activating a diverse array of defense mechanisms. Studies with these plants have provided a wealth of information on pathogen recognition, signal transduction and the activation of defense responses. However, very little is known about the infection and defense responses of the bryophyte, Physcomitrella patens, to well-studied phytopathogens. The purpose of this study was to determine: i) whether two representative broad host range pathogens, Erwinia carotovora ssp. carotovora (E.c. carotovora) and Botrytis cinerea (B. cinerea), could infect Physcomitrella, and ii) whether B. cinerea, elicitors of a harpin (HrpN) producing E.c. carotovora strain (SCC1) or a HrpN-negative strain (SCC3193), could cause disease symptoms and induce defense responses in Physcomitrella.ResultsB. cinerea and E.c. carotovora were found to readily infect Physcomitrella gametophytic tissues and cause disease symptoms. Treatments with B. cinerea spores or cell-free culture filtrates from E.c. carotovoraSCC1 (CF(SCC1)), resulted in disease development with severe maceration of Physcomitrella tissues, while CF(SCC3193) produced only mild maceration. Although increased cell death was observed with either the CFs or B. cinerea, the occurrence of cytoplasmic shrinkage was only visible in Evans blue stained protonemal cells treated with CF(SCC1) or inoculated with B. cinerea. Most cells showing cytoplasmic shrinkage accumulated autofluorescent compounds and brown chloroplasts were evident in a high proportion of these cells. CF treatments and B. cinerea inoculation induced the expression of the defense-related genes: PR-1, PAL, CHS and LOX.ConclusionB. cinerea and E.c. carotovora elicitors induce a defense response in Physcomitrella, as evidenced by enhanced expression of conserved plant defense-related genes. Since cytoplasmic shrinkage is the most common morphological change observed in plant PCD, and that harpins and B. cinerea induce this type of cell death in vascular plants, our results suggest that E.c. carotovora CFSCC1 containing HrpN and B. cinerea could also induce this type of cell death in Physcomitrella. Our studies thus establish Physcomitrella as an experimental host for investigation of plant-pathogen interactions and B. cinerea and elicitors of E.c. carotovora as promising tools for understanding the mechanisms involved in defense responses and in pathogen-mediated cell death in this simple land plant.

Pythium infection activates conserved plant defense responses in mosses.

The moss Physcomitrella patens (P. patens) is a useful model to study abiotic stress responses since it is highly tolerant to drought, salt and osmotic stress. However, very little is known about the defense mechanisms activated in this moss after pathogen assault. In this study, we show that P. patens activated multiple and similar responses against Pythium irregulare and Pythium debaryanum, including the reinforcement of the cell wall, induction of the defense genes CHS, LOX and PAL, and accumulation of the signaling molecules jasmonic acid (JA) and its precursor 12-oxo-phytodienoic acid (OPDA). However, theses responses were not sufficient and infection could not be prevented leading to hyphae colonization of moss tissues and plant decay. Pythium infection induced reactive oxygen species production and caused cell death of moss tissues. Taken together, these data indicate that Pythium infection activates in P. patens common responses to those previously characterized in flowering plants. Microscopic analysis also revealed intracellular relocation of chloroplasts in Pythium-infected tissues toward the infection site. In addition, OPDA, JA and its methyl ester methyl jasmonate induced the expression of PAL. Our results show for the first time JA and OPDA accumulation in a moss and suggest that this defense pathway is functional and has been maintained during the evolution of plants.

Activation of Defense Mechanisms against Pathogens in Mosses and Flowering Plants.

During evolution, plants have developed mechanisms to cope with and adapt to different types of stress, including microbial infection. Once the stress is sensed, signaling pathways are activated, leading to the induced expression of genes with different roles in defense. Mosses (Bryophytes) are non-vascular plants that diverged from flowering plants more than 450 million years ago, allowing comparative studies of the evolution of defense-related genes and defensive metabolites produced after microbial infection. The ancestral position among land plants, the sequenced genome and the feasibility of generating targeted knock-out mutants by homologous recombination has made the moss Physcomitrella patens an attractive model to perform functional studies of plant genes involved in stress responses. This paper reviews the current knowledge of inducible defense mechanisms in P. patens and compares them to those activated in flowering plants after pathogen assault, including the reinforcement of the cell wall, ROS production, programmed cell death, activation of defense genes and synthesis of secondary metabolites and defense hormones. The knowledge generated in P. patens together with comparative studies in flowering plants will help to identify key components in plant defense responses and to design novel strategies to enhance resistance to biotic stress.

Xanthomonas axonopodis pv. citri enters the VBNC state after copper treatment and retains its virulence

The most severe form of citrus canker disease is caused by Xanthomonas axonopodis pv. citri (Xac) and affects all types of important citrus crops, reducing fruit yield and quality. Copper-based products are routinely used as a standard control measure for citrus canker. In this work we demonstrate that copper treatment induces the viable but nonculturable (VBNC) state in Xac but does not prevent the development of symptoms in susceptible plants. Short-term exposures to different concentrations of copper solutions were assayed to determine which treatment resulted in Xac nonculturability. Treatment of 10(6) mL(-1) Xac cells for 10 min in a 135-muM CuSO(4) solution (equivalent to three times the free soluble copper concentration applied in one field treatment) resulted in nonculturability. However, 16% of cells were viable based on 5-cyano-2,3-ditolyl tetrazolium chloride staining and 1% were capable of producing canker lesions after infiltrating grapefruit plants. If induction of the VBNC state in Xac cells were to occur under field conditions, this would have to be taken into consideration for an effective control of canker disease.

Physcomitrella patens activates reinforcement of the cell wall, programmed cell death and accumulation of evolutionary conserved defence signals, such as salicylic acid and 12-oxo-phytodienoic acid, but not jasmonic acid, upon Botrytis cinerea infection

The moss Physcomitrella patens is an evolutionarily basal model system suitable for the analysis of plant defence responses activated after pathogen assault. Upon infection with the necrotroph Botrytis cinerea, several defence mechanisms are induced in P. patens, including the fortification of the plant cell wall by the incorporation of phenolic compounds and the induced expression of related genes. Botrytis cinerea infection also activates the accumulation of reactive oxygen species and cell death with hallmarks of programmed cell death in moss tissues. Salicylic acid (SA) levels also increase after fungal infection, and treatment with SA enhances transcript accumulation of the defence gene phenylalanine ammonia-lyase (PAL) in P. patens colonies. The expression levels of the genes involved in 12-oxo-phytodienoic acid (OPDA) synthesis, including lipoxygenase (LOX) and allene oxide synthase (AOS), increase in P. patens gametophytes after pathogen assault, together with a rise in free linolenic acid and OPDA concentrations. However, jasmonic acid (JA) could not be detected in healthy or infected tissues of this plant. Our results suggest that, although conserved defence signals, such as SA and OPDA, are synthesized and are probably involved in the defence response of P. patens against B. cinerea infection, JA production appears to be missing. Interestingly, P. patens responds to OPDA and methyl jasmonate by reducing moss colony growth and rhizoid length, suggesting that jasmonate perception is present in mosses. Thus, P. patens can provide clues with regard to the evolution of different defence pathways in plants, including signalling and perception of OPDA and jasmonates in nonflowering and flowering plants.

Functional Analysis of α-DOX2, an Active α-Dioxygenase Critical for Normal Development in Tomato Plants

Plant α-dioxygenases initiate the synthesis of oxylipins by catalyzing the incorporation of molecular oxygen at the α-methylene carbon atom of fatty acids. Previously, α-DOX1 has been shown to display α-dioxygenase activity and to be implicated in plant defense. In this study, we investigated the function of a second α-dioxygenase isoform, α-DOX2, in tomato (Solanum lycopersicum) and Arabidopsis (Arabidopsis thaliana). Recombinant Slα-DOX2 and Atα-DOX2 proteins catalyzed the conversion of a wide range of fatty acids into 2(R)-hydroperoxy derivatives. Expression of Slα-DOX2 and Atα-DOX2 was found in seedlings and increased during senescence induced by detachment of leaves. In contrast, microbial infection, earlier known to increase the expression of α-DOX1, did not alter the expression of Slα-DOX2 or Atα-DOX2. The tomato mutant divaricata, characterized by early dwarfing and anthocyanin accumulation, carries a mutation at the Slα-DOX2 locus and was chosen for functional studies of α-DOX2. Transcriptional changes in such mutants showed the up-regulation of genes playing roles in lipid and phenylpropanoid metabolism, the latter being in consonance with the anthocyanin accumulation. Transgenic expression of Atα-DOX2 and Slα-DOX2 in divaricata partially complemented the compromised phenotype in mature plants and fully complemented it in seedlings, thus indicating the functional exchangeability between α-DOX2 from tomato and Arabidopsis. However, deletion of Atα-DOX2 in Arabidopsis plants did not provoke any visible phenotypic alteration indicating that the relative importance of α-DOX2 in plant physiology is species specific.

Multiple defence signals induced by Erwinia carotovora ssp. carotovora elicitors in potato

SUMMARY Signal pathways involved in Solanum tuberosum-Erwinia carotovora ssp. carotovora(SCC3193) interaction were characterized. To this end, the concentration of several signal molecules implicated in plant defence such as ethylene (ET), jasmonates (JA) and salicylic acid (SA) were measured in potato plants treated by cell-free culture filtrates (CF) from E. c. carotovora(SCC3193). Furthermore, the presence of other potential signalling compounds such as cinnamic acid (CA) and related aromatic compounds was screened in the elicitor-treated plants. The activity of these signal compounds as inducers of defence-related genes such as drd-1 (a defence-related alcohol dehydrogenase), pinII (proteinase inhibitor II), chtB4 (basic chitinase) and chtA2 (acidic chitinase) was characterized. The results demonstrate that ET, JA and CA accumulate in potato tissues in response to CF. These signal molecules were shown to induce differential expression of drd-1, pinII, chtB4 and chtA2. Our data suggest that in addition to ET and JA, CA and possibly other aromatic compounds also may play a role in defence signalling in potato.

Toward a global database for the molecular typing of Saccharomyces cerevisiae strains

Most of the yeast strains used in fermented beverages and foods are classified as Saccharomyces cerevisiae. However, different strains are suitable for different fermentation processes. The purpose of this work is the proposal of a standardized methodology for the molecular genotyping of S. cerevisiae strains based on polymorphisms at microsatellite loci and/or single nucleotide polymorphisms (SNPs). Single nucleotide variants in the coding region of FLO8, a key regulator of flocculation and pseudohyphae formation, were analyzed in a subset of Uruguayan wine strains. Polymorphism analysis at nine microsatellite loci (selected from 33 loci tested) was performed in a collection of 120 strains, mostly wine strains, from different origins. From a total of 184 different alleles scored, 50 were exclusive alleles that could identify 29 strains. Four selected microsatellite loci are located within or near genes of putative enological interest. The Uruguayan strains are highly diverse and evenly distributed in the phylogenetic reconstructions, suggesting an evolutionary history previous to human use. The Saccharomyces cerevisiae Microsatellites and SNPs Genotyping Database is presented (www.pasteur.edu.uy/yeast). Comparison of standardized results from strains coming from different settings (industrial, clinical, environmental) will provide a reliable and growing source of information on the molecular biodiversity of S. cerevisiae strains.

Fatty acid α-dioxygenases

Abstract This paper will briefly review the biochemistry, molecular biology and functionality of α-dioxygenases from tobacco, Arabidopsis , cucumber, pea, rice and algae. α-Dioxygenase treated in this review has emerged as a third group of fatty acid dioxygenases along with lipoxygenases and endoperoxide synthases.

The Physcomitrella patens unique alpha-dioxygenase participates in both developmental processes and defense responses

BackgroundPlant α-dioxygenases catalyze the incorporation of molecular oxygen into polyunsaturated fatty acids leading to the formation of oxylipins. In flowering plants, two main groups of α-DOXs have been described. While the α-DOX1 isoforms are mainly involved in defense responses against microbial infection and herbivores, the α-DOX2 isoforms are mostly related to development. To gain insight into the roles played by these enzymes during land plant evolution, we performed biochemical, genetic and molecular analyses to examine the function of the single copy moss Physcomitrella patens α-DOX (Ppα-DOX) in development and defense against pathogens.ResultsRecombinant Ppα-DOX protein catalyzed the conversion of fatty acids into 2-hydroperoxy derivatives with a substrate preference for α-linolenic, linoleic and palmitic acids. Ppα-DOX is expressed during development in tips of young protonemal filaments with maximum expression levels in mitotically active undifferentiated apical cells. In leafy gametophores, Ppα-DOX is expressed in auxin producing tissues, including rhizoid and axillary hairs. Ppα-DOX transcript levels and Ppα-DOX activity increased in moss tissues infected with Botrytis cinerea or treated with Pectobacterium carotovorum elicitors. In B. cinerea infected leaves, Ppα-DOX-GUS proteins accumulated in cells surrounding infected cells, suggesting a protective mechanism. Targeted disruption of Ppα-DOX did not cause a visible developmental alteration and did not compromise the defense response. However, overexpressing Ppα-DOX, or incubating wild-type tissues with Ppα-DOX-derived oxylipins, principally the aldehyde heptadecatrienal, resulted in smaller moss colonies with less protonemal tissues, due to a reduction of caulonemal filament growth and a reduction of chloronemal cell size compared with normal tissues. In addition, Ppα-DOX overexpression and treatments with Ppα-DOX-derived oxylipins reduced cellular damage caused by elicitors of P. carotovorum.ConclusionsOur study shows that the unique α-DOX of the primitive land plant P. patens, although apparently not crucial, participates both in development and in the defense response against pathogens, suggesting that α-DOXs from flowering plants could have originated by duplication and successive functional diversification after the divergence from bryophytes.