Agrobacterium rhizogenes rolB oncogene: An intriguing player for many roles.

The rolB oncogene is one of the so-called rol genes found in the T-DNA region of the Agrobacterium rhizogenes Ri plasmid and involved in the hairy root syndrome, a tumour characterized by adventitious root overgrowth on plant stem. rolB produces in plants a peculiar phenotype that, together with its root-inducing capacity, has been connected to auxin sensitivity. The gene is able to modify the plant genetic programme to induce meristem cells and direct them to differentiate not only roots, but also other cells, tissues or organs. Besides its essential function in hairy root pathogenesis, the rolB role has been progressively extended to cover several physiological aspects in the transgenic plants: from secondary metabolites production and ROS inhibition, to abiotic and biotic stress tolerance and photosynthesis improvement. Some of the observed effects could be determined, at least in part, through microRNAs molecules, suggesting an epigenetic control rolB-mediated. These multifaceted capacities could allow plants to withstand adverse environmental conditions, enhancing fitness. In spite of this expanding knowledge, functional analyses did not detect yet any definitive rolB-derived biochemical product, even if more than one enzymatic activity has been ascribed to it. Moreover, phylogenetic and evolutionary studies evidenced no homology with any plant sequences but, otherwise, it belongs to the Plast family, a group of rolB-homologous bacterial genes. Finally, the finding of sequences similar to rolB in plants not infected by A. rhizogenes suggests a hypothetical plant origin for this gene, implying different possibilities about its evolution.

Effect of far-red light exposure on photosynthesis and photoprotection in tomato plants transgenic for the Agrobacterium rhizogenes rolB gene.

Previous work showed in tomato plants harbouring the Agrobacterium rhizogenes rolB gene overexpression of genes involved in chloroplast function and stress response, significant increase in non-photochemical quenching and chlorophyll a and b content, and reduced chlorophyll a/b ratio. The latter condition being typical of plant shade where far-red is dominant, suggested a role for rolB in improving photosynthesis in such condition. To gain a better insight into these results, the photosynthetic performance of transgenic and control plants was compared by means of variable fluorescence kinetics with a WATER-PAM chlorophyll fluorometer, after 6 days-exposure to white light and to a far-red-enriched light source. Photosynthetic parameters analysed were quantum yield of photosystem II photochemistry Y(II); qL, corresponding to the fraction of open PSII reaction centers in a "lake" model of photosystem II; non-photochemical quenching and Y(NO), describing, respectively, regulated and non-regulated pathways for dissipation of excess energy. Chlorophyll a and b content was also analysed by HPLC. Finally, real-time PCR was performed to quantify the expression level of some of the chloroplast-related genes already shown to be overexpressed in transgenic plants. Quantum yield of photosystem II photochemistry decreased with increasing light intensity, showing no significant differences in both plant genotypes and light regimen. qL, on the other hand, was significantly higher at low PAR intensities, in particular in FR-treated transgenic plants. Fate of remaining light energy, channelled into regulated or non-regulated dissipation pathways, was different in transgenic and control plants, indicating a higher capability for protection from photodamage in rolB plants, particularly after exposure to far-red-enriched light. Chlorophyll a/b ratio was also decreased in transgenic plants under far-red-enriched light with respect to white light. Finally, qPCR showed that the expression of genes encoding small heat shock protein, chlorophyll a/b binding protein and carbonic anhydrase was significantly induced by far-red-enriched condition. Taken together, these data suggest the involvement of rolB in photosynthesis modulation under far-red-rich light in tomato.

Geosmithia-Ophiostoma: a New Fungus-Fungus Association.

In Europe as in North America, elms are devastated by Dutch elm disease (DED), caused by the alien ascomycete Ophiostoma novo-ulmi. Pathogen dispersal and transmission are ensured by local species of bark beetles, which established a novel association with the fungus. Elm bark beetles also transport the Geosmithia fungi genus that is found in scolytids' galleries colonized by O. novo-ulmi. Widespread horizontal gene transfer between O. novo-ulmi and Geosmithia was recently observed. In order to define the relation between these two fungi in the DED pathosystem, O. novo-ulmi and Geosmithia species from elm, including a GFP-tagged strain, were grown in dual culture and mycelial interactions were observed by light and fluorescence microscopy. Growth and sporulation of O. novo-ulmi in the absence or presence of Geosmithia were compared. The impact of Geosmithia on DED severity was tested in vivo by co-inoculating Geosmithia and O. novo-ulmi in elms. A close and stable relation was observed between the two fungi, which may be classified as mycoparasitism by Geosmithia on O. novo-ulmi. These results prove the existence of a new component in the complex of organisms involved in DED, which might be capable of reducing the disease impact.

Correction to: Geosmithia-Ophiostoma: a New Fungus-Fungus Association.

The article Geosmithia-Ophiostoma: a New Fungus-Fungus Association, written by Alessia L. Pepori, Priscilla P. Bettini, Cecilia Comparini, Sabrina Sarrocco, Anna Bonini, Arcangela Frascella, Luisa Ghelardini, & Aniello Scala, Giovanni Vannacci, Alberto Santini.

Agrobacterium rhizogenes rolB gene affects photosynthesis and chlorophyll content in transgenic tomato (Solanum lycopersicum L.) plants.

Insertion of Agrobacterium rhizogenes rolB gene into plant genome affects plant development, hormone balance and defence. However, beside the current research, the overall transcriptional response and gene expression of rolB as a modulator in plant is unknown. Transformed rolB tomato plant (Solanum lycopersicum L.) cultivar Tondino has been used to investigate the differential expression profile. Tomato is a well-known model organism both at the genetic and molecular level, and one of the most important commercial food crops in the world. Through the construction and characterization of a cDNA subtracted library, we have investigated the differential gene expression between transgenic clones of rolB and control tomato and have evaluated genes specifically transcribed in transgenic rolB plants. Among the selected genes, five genes encoding for chlorophyll a/b binding protein, carbonic anhydrase, cytochrome b6/f complex Fe-S subunit, potassium efflux antiporter 3, and chloroplast small heat-shock protein, all involved in chloroplast function, were identified. Measurement of photosynthesis efficiency by the level of three different photosynthetic parameters (Fv/Fm, rETR, NPQ) showed rolB significant increase in non-photochemical quenching and a, b chlorophyll content. Our results point to highlight the role of rolB on plant fitness by improving photosynthesis.

Morphological and molecular characterisation of Geosmithia species on European elms.

Species of the genus Geosmithia are associated with insect species, mainly bark beetles. On Ulmus spp., the same beetles are also vectors of Ophiostoma ulmi s.l., the agent of Dutch elm disease (DED), a worldwide elm disease. Aim of this paper is to characterise Geosmithia species associated with elms and/or elm beetles in Europe. Seventy-two strains representative of all morphological taxonomic units were used to build a phylogenetic tree based on ITS, ß-tubulin and elongation factor 1-α gene regions. On the basis of molecular and morpho-physiological traits, seven taxonomic entities were identified. In addition to the species previously known our results assigned strains previously identified as Geosmithia pallida to two separate taxa: Geosmithia sp. 2 and Geosmithia sp. 5. Two new species, Geosmithia omnicola and Geosmithia ulmacea, are described. Two strains were assigned to the partially described species Geosmithia sp. 20. Geosmithia species living on Ulmus do not discriminate between elm species, but between different environments. The association between Ulmus and Geosmithia is common, stable, and seems to be related to specific vectors. The relationship between Geosmithia and Ophiostoma would deserve further investigation, as these fungi share the same vectors and habitat for a significant part of their life cycles.

Interspecific variability of class II hydrophobin GEO1 in the genus Geosmithia.

The genus Geosmithia Pitt (Ascomycota: Hypocreales) comprises cosmopolite fungi living in the galleries built by phloeophagous insects. Following the characterization in Geosmithia species 5 of the class II hydrophobin GEO1 and of the corresponding gene, the presence of the geo1 gene was investigated in 26 strains derived from different host plants and geographic locations and representing the whole phylogenetic diversity of the genus. The geo1 gene was detected in all the species tested where it maintained the general organization shown in Geosmithia species 5, comprising three exons and two introns. Size variations were found in both introns and in the first exon, the latter being due to the presence of an intragenic tandem repeat sequence corresponding to a stretch of glycine residues in the deduced proteins. At the amino acid level the deduced proteins had 44.6 % identity and no major differences in the biochemical parameters (pI, GRAVY index, hydropathy plots) were found. GEO1 release in the fungal culture medium was also assessed by turbidimetric assay and SDS-PAGE, and showed high variability between species. The phylogeny based on the geo1 sequences did not correspond to that generated from a neutral marker (ITS rDNA), suggesting that sequence similarities could be influenced by other factors than phylogenetic relatedness, such as the intimacy of the symbiosis with insect vectors. The hypothesis of a strong selection pressure on the geo1 gene was sustained by the low values (<1) of non synonymous to synonymous nucleotide substitutions ratios (Ka/Ks), which suggest that purifying selection might act on this gene. These results are compatible with either a birth-and-death evolution scenario or horizontal transfer of the gene between Geosmithia species.

Widespread horizontal transfer of the cerato-ulmin gene between Ophiostoma novo-ulmi and Geosmithia species.

Previous work had shown that a sequence homologous to the gene encoding class II hydrophobin cerato-ulmin from the fungus Ophiostoma novo-ulmi, the causal agent of Dutch Elm Disease (DED), was present in a strain of the unrelated species Geosmithia species 5 (Ascomycota: Hypocreales) isolated from Ulmus minor affected by DED. As both fungi occupy the same habitat, even if different ecological niches, the occurrence of horizontal gene transfer was proposed. In the present work we have analysed for the presence of the cerato-ulmin gene 70 Geosmithia strains representing 29 species, isolated from different host plants and geographic locations. The gene was found in 52.1 % of the strains derived from elm trees, while none of those isolated from nonelms possessed it. The expression of the gene in Geosmithia was also assessed by real time PCR in different growth conditions (liquid culture, solid culture, elm sawdust, dual culture with O. novo-ulmi), and was found to be extremely low in all conditions tested. On the basis of these results we propose that the cerato-ulmin gene is not functional in Geosmithia, but can be considered instead a marker of more extensive transfers of genetic material as shown in other fungi.

Identification and characterization of GEO1, a new class II hydrophobin from Geosmithia spp.

In the present paper we describe a new noncatalytic protein belonging to the hydrophobin family, designated GEO1, purified from the culture filtrate of Geosmithia pallida (Ascomycota: Hypocreales), and the corresponding gene sequence. In the fungal genome, GEO1 was encoded by a single-copy gene with a 450 bp open reading frame interrupted by 2 small introns whose primary translation product was 109 amino acids long and included a 23 amino acids signal peptide. The mature protein had a molecular mass of 8111.75 Da and a theoretical pI of 4.33. The deduced amino acid sequence showed similarity to class II hydrophobins and contained 8 conserved cysteine residues, present in all hydrophobins isolated so far. Biochemical properties, such as foam-forming ability and trapezoid-like shape of a GEO1 drop, also resembled the typical features of the class II hydrophobins. Expression of the geo1 gene was assessed after 2, 4, 7, 9, and 11 days of culture and showed that the geo1 transcript appeared after 7 days and increased up to 11 days.

The expression of the cerato-platanin gene is related to hyphal growth and chlamydospores formation in Ceratocystis platani.

Cerato-platanin (CP) is a protein produced by Ceratocystis platani, the causal agent of canker stain disease of plane trees. CP is the first member of the 'cerato-platanin family', and its role as a pathogen-associated molecular pattern (PAMP), inducing defence responses both in host and nonhost plants, is established. However, the primary role of CP and its homologues in the fungal life remains unknown. In the present work, we investigated the regulation of the cp gene during the in vitro growth of C. platani in different conditions and under the effect of potential stress factors. Fungal growth and conidiogenesis were also analysed. Results showed that cp is a single-copy gene whose expression level is strictly associated with hyphal growth and with chlamydospores formation. The analysis of a 1368 bp 5'-flanking region revealed putative motifs that could be involved in the regulation of gene expression in response to stress and developmental cues. Taking into account the localization of CP in the fungal cell wall and the recently published 3D structure of the protein, our results support a role for CP in growth and developmental processes of C. platani.

Isolation of the orthologue of the cerato-ulmin gene in Ophiostoma quercus and characterization of the purified protein.

Ophiostoma quercus is an ophiostomatoid fungus strictly related to the Ophiostoma's (O. ulmi, O. novo-ulmi, and O. himal-ulmi) that cause Dutch elm disease (DED). O. quercus has a number of morphological characteristics in common with the DED pathogens, and is a well-known and economically important sapstaining fungus occurring worldwide on hardwoods and commercially produced pines, and causes typical cankers on oak stems. In elm trees O. quercus can survive for months without causing any disease symptoms. DED fungi produce cerato-ulmin (CU), a class II hydrophobin, which is generally considered as the main toxin potentially involved in various phases of the DED pathogenesis. In the present work we isolated and sequenced the orthologue of the cu gene in the O. quercus isolates H988, H1042, and H2053. Moreover the CU protein from O. quercus isolate H988 was also purified and characterized. Sequence analysis showed that there is a pronounced difference between the whole cu gene region of O. quercus and the homologous fragments of the DED-causing species O. ulmi, O. novo-ulmi, and O. himal-ulmi. It also appeared that differences in the structural conformation of the promoter were unlikely to play a role in the modulation of the transcript level and that, for O. quercus, differences in CU production did not result from the potential different regulation levels. Clear differences were shown in the transcriptional unit of the cu genes and in the amino acid sequences among all the CUs. The purified O. quercus CU was separated using matrix-assisted laser desorption ionization/time of flight (MALDI-TOF) spectrometry into seven forms of increasing molecular weight from 7190 to 7724Da. The hydrophobicity profiles indicated that two regions of the O. quercus CU protein were more hydrophobic than the corresponding regions of the CUs of the DED fungi. The O. quercus CUs had theoretical isoelectric point values similar to those of the DED fungi. Finally, the contradiction between the consistent differences between these four Ophiostoma species in the cu gene region and in the CU proteins and their strict phylogenetic relationship is discussed.

Identification by suppression subtractive hybridization of genes expressed in pear (Pyrus spp.) upon infestation with Cacopsylla pyri (Homoptera: Psyllidae).

The molecular interaction between pear tree (Pyrus spp.) and the phloem-feeding psylla Cacopsylla pyri (Linnaeus) was investigated through the construction and characterization of cDNA subtracted libraries. Genes expressed upon insect infestation were identified in the susceptible pear cultivar Bartlett and in the resistant selection NY10355. In both interactions, genes involved in the plant defense response were induced, confirming the observed similarity between the response to pathogens and to insects with piercing/sucking mouthparts. However, the two expression profiles were found to be different, with more genes involved in the response to biotic and abiotic stress being activated in the resistant plant than in the susceptible one. Further characterization of the identified genes could lead to the development of molecular markers associated with tolerance/resistance to pear psylla.