Graciela L. Salerno

ISSR markers detect high genetic variation among Fusarium poae isolates from Argentina and England

Fusarium poae is one of the Fusarium species isolated from cereal grains infected by Fusarium head blight (FHB), and in recent years it has been identified as a major FHB component. In this study, 97 F. poae isolates from Argentina (n = 62) and England (n = 35) were analysed by inter-simple sequence repeats (ISSR) to examine the genetic diversity and to determine whether intraspecific variation could be correlated with geographic and/or host origin. The molecular analysis showed high intraspecific variability within F. poae isolates, but did not reveal a clear relationship between variability and the host/geographic origin. Fusarium poae isolates from the same geographic region or host appeared in different subclusters. Conversely, isolates with the same haplotype were also collected from different geographic regions. However, we did observe subclusters consisting of isolates from Argentina only or from England only. Furthermore, a single seed sample was found to host different haplotypes. Analysis of molecular variance (AMOVA) indicated a high genetic variability in F. poae, with most of the genetic variability explained by differences within, rather than between Argentinean and English populations. This is the first report on genetic diversity of F. poae using ISSR markers. Moreover, ISSR fingerprinting generates highly polymorphic markers for F. poae and proved to be a useful and reliable assay for genetic variability studies.

The unique nucleotide specificity of the sucrose synthase from Thermosynechococcus elongatus

Sucrose synthase catalyzes the reversible conversion of sucrose and UDP into fructose and UDP‐glucose. In filamentous cyanobacteria, the sucrose cleavage direction plays a key physiological function in carbon metabolism, nitrogen fixation, and stress tolerance. In unicellular strains, the function of sucrose synthase has not been elucidated. We report a detailed biochemical characterization of sucrose synthase from Thermosynechococcus elongatus after the gene was artificially synthesized for optimal expression in Escherichia coli. The homogeneous recombinant sucrose synthase was highly specific for ADP as substrate, constituting the first one with this unique characteristic, and strongly suggesting an interaction between sucrose and glycogen metabolism.

A mitochondrial alkaline/neutral invertase isoform (A/N-InvC) functions in developmental energy-demanding processes in Arabidopsis

Recent findings demonstrate that alkaline/neutral invertases (A/N-Invs), enzymes that catalyze the breakdown of sucrose into glucose and fructose, are essential proteins in plant life. The fact that different isoforms are present in multiple locations makes them candidates for the coordination of metabolic processes. In the present study, we functionally characterized the encoding gene of a novel A/N-Inv (named A/N-InvC) from Arabidopsis, which localizes in mitochondria. A/N-InvC is expressed in roots, in aerial parts (shoots and leaves) and flowers. A detailed phenotypic analysis of knockout mutant plants (invc) reveals an impaired growth phenotype. Shoot growth was severely reduced, but root development was not affected as reported for A/N-InvA mutant (inva) plants. Remarkably, germination and flowering, two energy demanding processes, were the most affected stages. The effect of exogenous growth regulators led us to suggest that A/N-InvC may be modulating hormone balance in relation to the radicle emergence. We also show that oxygen consumption is reduced in inva and invc in comparison with wild-type plants, indicating that both organelle isoenzymes may play a fundamental role in mitochondrion functionality. Taken together, our results emphasize the involvement of mitochondrial A/N-Invs in developmental processes and uncover the possibility of playing different roles for the two isoforms located in the organelle.

Phylogenetic relationships of Fusarium poae based on EF-1α and mtSSU sequences

A molecular phylogenetic analysis of Fusarium poae isolates from South America (Argentina) and Europe (mainly England, Germany, Italy) was performed using 98 F. poae, four Fusarium culmorum, two Fusarium sporotrichioides and one Fusarium langsethiae isolates. Phylogenetic analyses were performed using nuclear (translation elongation factor 1-alpha, EF-1 alpha) and mitochondrial (mitochondrial small subunit rDNA, mtSSU) sequences. Partitioned (each dataset separately) and combined (EF-1 alpha+mtSSU) analyses did not reveal any clear correlations from the inferred branching topology, between the distribution of observed haplotypes and the geographic origin and/or host species. Results from the present study confirmed that isolates from F. poae form a monophyletic group, and the low variability within isolates from a broad geographic range suggests a common lineage history. Among F. poae isolates from Argentina, however, some were found to possess an insert within mtSSU with structural similarities to group IC2 introns. F. poae isolates differing by the presence/absence of a mtSSU insertion were characterized further by analysis of a portion of the Tri5 gene, but this sequence was unable to reveal variability. The presence of this insert only within isolates from Argentina suggests that evolutionary events (insertions/deletions) are probably taking place within the Argentinian F. poae isolates, and that the acquisition of this insert occurred after geographic isolation of the Argentinian and European populations.

Functional characterization of Synechococcus amylosucrase and fructokinase encoding genes discovers two novel actors on the stage of cyanobacterial sucrose metabolism.

Plants and most cyanobacteria metabolize sucrose (Suc) with a similar set of enzymes. In Synechococcus sp. PCC 7002, a marine cyanobacterium strain, genes involved in Suc synthesis (spsA and sppA) have been characterized; however, its breakdown was still unknown. Indeed, neither invertase nor sucrose synthase genes, usually found in plants and cyanobacteria, were found in that Synechococcus genome. In the present study, we functionally characterized the amsA gene that codes for an amylosucrase (AMS), a glycoside-hydrolase family 13 enzyme described in bacteria, which may catabolyze Suc in Synechococcus. Additionally, we identified and characterized the frkA gene that codes for a fructokinase (FRK), enzyme that yields fructose-6P, one of the substrates for Suc synthesis. Interestingly, we demonstrate that spsA, sppA, frkA and amsA are grouped in a transcriptional unit that were named Suc cluster, whose expression is increased in response to a salt treatment. This is the first report on the characterization of an AMS and FRK in an oxygenic photosynthetic microorganism, which could be associated with Suc metabolism.

Sublethal effects of acaricides and Nosema ceranae infection on immune related gene expression in honeybees

Nosema ceranae is an obligate intracellular parasite and the etiologic agent of Nosemosis that affects honeybees. Beside the stress caused by this pathogen, honeybee colonies are exposed to pesticides under beekeeper intervention, such as acaricides to control Varroa mites. These compounds can accumulate at high concentrations in apicultural matrices. In this work, the effects of parasitosis/acaricide on genes involved in honeybee immunity and survival were evaluated. Nurse bees were infected with N. ceranae and/or were chronically treated with sublethal doses of coumaphos or tau-fluvalinate, the two most abundant pesticides recorded in productive hives. Our results demonstrate the following: (1) honeybee survival was not affected by any of the treatments; (2) parasite development was not altered by acaricide treatments; (3) coumaphos exposure decreased lysozyme expression; (4) N. ceranae reduced levels of vitellogenin transcripts independently of the presence of acaricides. However, combined effects among stressors on imagoes were not recorded. Sublethal doses of acaricides and their interaction with other ubiquitous parasites in colonies, extending the experimental time, are of particular interest in further research work.

The polyphasic analysis of two native Raphidiopsis isolates supports the unification of the genera Raphidiopsis and Cylindrospermopsis (Nostocales, Cyanobacteria)

Abstract: Raphidiopsis and Cylindrospermopsis are planktic, freshwater bloom-forming cyanobacteria of great concern to human health due to the production of potent cyanotoxins. The presence (in Cylindrospermopsis) or absence (in Raphidiopsis) of heterocytes is the traditional character used to distinguish them. This has led to misidentifications and to questions about the validity of the genus Raphidiopsis. We studied two strains of R. mediterranea isolated from Argentinean shallow lakes using a polyphasic approach that included the morphological description of the natural populations and their ultrastructural, physiological and molecular characterisation. Heterocyte differentiation was not observed in the field or in cultures of R. mediterranea submitted to nitrogen deprivation. These results support the occurrence of stable native populations of R. mediterranea without heterocytes, which would not be a part of the Cylindrospermopsis complex life cycle. Based on 16S rRNA, 16S–23S ITS, and cpcBA-IGS sequences, these two genera are virtually identical. Thus, strains of Raphidiopsis and Cylindrospermopsis make up a monophyletic lineage in all phylogenetic reconstructions. Furthermore, the 16S–23S ITS secondary structure provided further evidence that these two genera cannot be separated. The intermixed position in the trees points to several losses of heterocytes during the evolution of these cyanobacteria. We conclude that these two genera should not be regarded as separate and distinct generic units and propose their unification under the name Raphidiopsis, respecting the principle of priority. Accordingly, we revisited and emended the description of Raphidiopsis.

Sucrose in bloom‐forming cyanobacteria: loss and gain of genes involved in its biosynthesis

Bloom-forming cyanobacteria are widely distributed in freshwater ecosystems. To cope with salinity fluctuations, cyanobacteria synthesize compatible solutes, such as sucrose, to maintain the intracellular osmotic balance. The screening of cyanobacterial genomes revealed that homologues to sucrose metabolism-related genes only occur in few bloom-forming strains, mostly belonging to Nostocales and Stigonematales orders. Remarkably, among Chroococcales and Oscillatoriales strains, homologues were only found in M. aeruginosa PCC 7806 and Leptolyngbya boryana PCC 6306, suggesting a massive loss of sucrose metabolism in bloom-forming strains of these orders. After a complete functional characterization of sucrose genes in M. aeruginosa PCC 7806, we showed that sucrose metabolism depends on the expression of a gene cluster that defines a transcriptional unit, unique among all sucrose-containing cyanobacteria. It was also demonstrated that the expression of the encoding genes of sucrose-related proteins is stimulated by salt. In view of its ancestral origin in cyanobacteria, the fact that most bloom-forming strains lack sucrose metabolism indicates that the genes involved might have been lost during evolution. However, in a particular strain, like M. aeruginosa PCC 7806, sucrose synthesis genes were probably regained by horizontal gene transfer, which could be hypothesized as a response to salinity fluctuations.

Genetic diversity of Fusarium graminearum sensu lato isolates from wheat associated with Fusarium Head Blight in diverse geographic locations of Argentina

Fusarium Head Blight is an important wheat disease in the Argentine Pampas region, being Fusarium graminearum the predominant pathogen. DNA polymorphism of the isolates was analyzed by IGS-RFLP and ISSR. IGS-RFLP and ISSR profiling were carried out using six endonucleases and eight primers, respectively. IGS-RFLP yielded 41 bands, 30 of which were polymorphic while ISSR produced 87 bands with 47 polymorphic bands. Both markers showed genetic variability among the analyzed isolates; however, IGS-RFLP was more efficient than ISSR, showing a higher polymorphic average (59.91%) than the latter (44.11%). The averages of polymorphic information content (PIC) were 0.211 and 0.129, respectively. Twenty haplotypes were identified by IGS-RFLP and 15 haplotypes by ISSR. Genotype clustering within dendrograms was different for both types of markers. The genetic groups obtained by IGS-RFLP showed a partial association to geographic origin. This is the first report on genetic variability of F. graminearum isolates from wheat in Argentina using IGS-RFLP and ISSR markers.

The riddle of mitochondrial alkaline/neutral invertases: A novel Arabidopsis isoform mainly present in reproductive tissues and involved in root ROS production

Alkaline/neutral invertases (A/N-Inv), glucosidases that irreversibly hydrolyze sucrose into glucose and fructose, play significant roles in plant growth, development, and stress adaptation. They occur as multiple isoforms located in the cytosol or organelles. In Arabidopsis thaliana, two mitochondrial A/N-Inv genes (A/N-InvA and A/N-InvC) have already been investigated. In this study, we functionally characterized A/N-InvH, a third Arabidopsis gene coding for a mitochondrial-targeted protein. The phenotypic analysis of knockout mutant plants (invh) showed a severely reduced shoot growth, while root development was not affected. The emergence of the first floral bud and the opening of the first flower were the most affected stages, presenting a significant delay. A/N-InvH transcription is markedly active in reproductive tissues. It is also expressed in the elongation and apical meristem root zones. Our results show that A/N-InvH expression is not evident in photosynthetic tissues, despite being of relevance in developmental processes and mitochondrial functional status. NaCl and mannitol treatments increased A/N-InvH expression twofold in the columella root cap. Moreover, the absence of A/N-InvH prevented ROS formation, not only in invh roots of salt- and ABA-treated seedlings but also in invh control roots. We hypothesize that this isoform may take part in the ROS/sugar (sucrose or its hydrolysis products) signaling pathway network, involved in reproductive tissue development, cell elongation, and abiotic stress responses.