Filippos Ververidis

Metabolic engineering of the complete pathway leading to heterologous biosynthesis of various flavonoids and stilbenoids in Saccharomyces cerevisiae

Chemical or biological synthesis of plant secondary metabolites has attracted increasing interest due to their proven or assumed beneficial properties and health promoting effects. Resveratrol, a stilbenoid, naringenin, a flavanone, genistein, an isoflavone, and the flavonols kaempferol and quercetin have been shown to possess high nutritional and agricultural value. Four metabolically engineered yeast strains harboring plasmids with heterologous genes for enzymes involved in the biosynthesis of these compounds from phenylalanine have been constructed. Time course analyses of precursor utilization and end-product accumulation were carried out establishing the production of 0.29-0.31 mg/L of trans-resveratrol, 8.9-15.6 mg/L of naringenin, 0.1-7.7 mg/L of genistein, 0.9-4.6 mg/L of kaempferol and 0.26-0.38 mg/L of quercetin in defined media under optimal growth conditions. The recombinant yeast strains can be used further for the construction of improved flavonoid- and stilbenoid-overproducers.

Sensitive cells: enabling tools for static and dynamic control of microbial metabolic pathways.

Natural metabolic pathways are dynamically regulated at the transcriptional, translational, and protein levels. Despite this, traditional pathway engineering has relied on static control strategies to engender changes in metabolism, most likely due to ease of implementation and perceived predictability of design outcome. Increasingly in recent years, however, metabolic engineers have drawn inspiration from natural systems and have begun to harness dynamically controlled regulatory machinery to improve design of engineered microorganisms for production of specialty and commodity chemicals. Here, we review recent enabling technologies for engineering static control over pathway expression levels, and we discuss state-of-the-art dynamic control strategies that have yielded improved outcomes in the field of microbial metabolic engineering. Furthermore, we emphasize design of a novel class of genetically encoded controllers that will facilitate automatic, transient tuning of synthetic and endogenous pathways.

Comparative Genomics of Multiple Strains of Pseudomonas cannabina pv. alisalensis, a Potential Model Pathogen of Both Monocots and Dicots

Comparative genomics of closely related pathogens that differ in host range can provide insights into mechanisms of host-pathogen interactions and host adaptation. Furthermore, sequencing of multiple strains with the same host range reveals information concerning pathogen diversity and the molecular basis of virulence. Here we present a comparative analysis of draft genome sequences for four strains of Pseudomonas cannabina pathovar alisalensis (Pcal), which is pathogenic on a range of monocotyledonous and dicotyledonous plants. These draft genome sequences provide a foundation for understanding host range evolution across the monocot-dicot divide. Like other phytopathogenic pseudomonads, Pcal strains harboured a hrp/hrc gene cluster that codes for a type III secretion system. Phylogenetic analysis based on the hrp/hrc cluster genes/proteins, suggests localized recombination and functional divergence within the hrp/hrc cluster. Despite significant conservation of overall genetic content across Pcal genomes, comparison of type III effector repertoires reinforced previous molecular data suggesting the existence of two distinct lineages within this pathovar. Furthermore, all Pcal strains analyzed harbored two distinct genomic islands predicted to code for type VI secretion systems (T6SSs). While one of these systems was orthologous to known P. syringae T6SSs, the other more closely resembled a T6SS found within P. aeruginosa. In summary, our study provides a foundation to unravel Pcal adaptation to both monocot and dicot hosts and provides genetic insights into the mechanisms underlying pathogenicity.

Pseudomonas viridiflava , a Multi Host Plant Pathogen with Significant Genetic Variation at the Molecular Level

The pectinolytic species Pseudomonas viridiflava has a wide host range among plants, causing foliar and stem necrotic lesions and basal stem and root rots. However, little is known about the molecular evolution of this species. In this study we investigated the intraspecies genetic variation of P. viridiflava amongst local (Cretan), as well as international isolates of the pathogen. The genetic and phenotypic variability were investigated by molecular fingerprinting (rep-PCR) and partial sequencing of three housekeeping genes (gyrB, rpoD and rpoB), and by biochemical and pathogenicity profiling. The biochemical tests and pathogenicity profiling did not reveal any variability among the isolates studied. However, the molecular fingerprinting patterns and housekeeping gene sequences clearly differentiated them. In a broader phylogenetic comparison of housekeeping gene sequences deposited in GenBank, significant genetic variability at the molecular level was found between isolates of P. viridiflava originated from different host species as well as among isolates from the same host. Our results provide a basis for more comprehensive understanding of the biology, sources and shifts in genetic diversity and evolution of P. viridiflava populations and should support the development of molecular identification tools and epidemiological studies in diseases caused by this species.

Comparative genomic analysis of multiple strains of two unusual plant pathogens: Pseudomonas corrugata and Pseudomonas mediterranea

The non-fluorescent pseudomonads, Pseudomonas corrugata (Pcor) and P. mediterranea (Pmed), are closely related species that cause pith necrosis, a disease of tomato that causes severe crop losses. However, they also show strong antagonistic effects against economically important pathogens, demonstrating their potential for utilization as biological control agents. In addition, their metabolic versatility makes them attractive for the production of commercial biomolecules and bioremediation. An extensive comparative genomics study is required to dissect the mechanisms that Pcor and Pmed employ to cause disease, prevent disease caused by other pathogens, and to mine their genomes for genes that encode proteins involved in commercially important chemical pathways. Here, we present the draft genomes of nine Pcor and Pmed strains from different geographical locations. This analysis covered significant genetic heterogeneity and allowed in-depth genomic comparison. All examined strains were able to trigger symptoms in tomato plants but not all induced a hypersensitive-like response in Nicotiana benthamiana. Genome-mining revealed the absence of type III secretion system and known type III effector-encoding genes from all examined Pcor and Pmed strains. The lack of a type III secretion system appears to be unique among the plant pathogenic pseudomonads. Several gene clusters coding for type VI secretion system were detected in all genomes. Genome-mining also revealed the presence of gene clusters for biosynthesis of siderophores, polyketides, non-ribosomal peptides, and hydrogen cyanide. A highly conserved quorum sensing system was detected in all strains, although species specific differences were observed. Our study provides the basis for in-depth investigations regarding the molecular mechanisms underlying virulence strategies in the battle between plants and microbes.

A new genomovar of Pseudomonas cichorii, a causal agent of tomato pith necrosis

Recent taxonomic advances, based on biochemical and genotypic processes demonstrate that the plant pathogenic species Pseudomonas cichorii consists of a cluster of closely related genomic groups. Prior to this study, three morphotype groups had been described (C1-C3), all sharing various phenotypic and biochemical characters but partially differing in their DNA content. All entities of the complex could cause disease among a variety of hosts, including lettuce, celery, chrysanthemum and others. In this study, we present the biochemical and molecular characterization of P. cichorii isolates as the causal agent of pith necrosis of tomato plants. A detailed characterization of the genetic variability among strains belonging to P. cichorii was achieved using BOX-PCR and Multi Locus Sequence Analysis utilizing three housekeeping genes (gyrB, rpoD, rpoB). In addition, a number of biochemical and physiological tests were also used for the identification of the tomato P. cichorii isolates. To our knowledge, this is the first complete biochemical, molecular and phylogenetic study of P. cichorii strains isolated from tomato plants affected by pith necrosis disease. Our findings demonstrate the emergence of a new genomovar of P. cichorii, yet another indication for the genetic heterogeneity of the species.

First Report of Pseudomonas viridiflava Causing a Bacterial Blight of Artichoke Bract Leaves

In 2006, a disease was observed on two artichoke (Cynara scolymus L. cv. Lardati) fields in Crete, Greece, covering ~2 ha. Symptoms developed after several days of rainy and windy weather and >70% of capitula were affected, resulting in unmarketable produce. Initial symptoms were water-soaked, dark green spots on bracts with many sunken, necrotic, often elongated lesions, each with a brown-black center and surrounded by a water-soaked halo with a dark red-brown margin. Symptoms were more severe on inner bracts. Isolations from symptomatic, surface-disinfected bracts onto Kings B agar medium (KB) consistently yielded yellow bacterial colonies that produced a green-blue fluorescent pigment. Ten selected artichoke isolates, all gram-negative, presented the LOPAT profile (- - + - +) and were levan negative, oxidase negative, potato rot positive, arginine dihydrolase negative, and showed tobacco hypersensitive reaction. All isolates used L-arabinose, D(-)-tartrate, and L-lactate, but not sucrose, L(+)-tartrate, or trigonelline. Results were identical to those obtained with the reference strain of Pseudomonas viridiflava NCPPB 1249 (3), and strains PV3005 and PV3006 from eggplant (1). Based on these biochemical tests, 10 isolates were identified as P. viridiflava group II members of the LOPAT determinative scheme of Lelliott (1,2). Two artichoke isolates (PV608 and PV609) were selected for molecular characterization. The identity and phylogenetic analysis were determined by multilocus sequence typing with the gyrB, rpoD, and rpoB genes (PV608 Accession Nos. JN383375, JN383363, and JQ267546; PV609 Accession Nos. JN383376, JN383364, and JQ267547). BLAST searches showed highest nucleotide sequence identity (96%) with GenBank sequences of P. viridiflava reference strains NCPPB 963 and CFBP 2107. Pathogenicity of 10 artichoke isolates and reference strains was tested twice on detached capitulum bracts of artichoke cv. Lardati, as well as 4-week-old tomato plants of cv. ACE, and Chrysanthemum indicum cv. Reagan plants. Each isolate was inoculated onto 10 bracts by placing 15 μl of bacterial suspension (5 × 106 CFU/ml) of a 48-h culture in KB broth on the surface of the bract, and pricking the bract through the drop of bacterial suspension with a sterile needle. Each isolate was also inoculated onto five tomato and five chrysanthemum plants by dipping a sterile toothpick in the appropriate bacterial culture and pricking the surface of the stem. Ten control plants were inoculated similarly with sterile, distilled water. Inoculated bracts and plants were kept in boxes lined with moist filter paper at 25 to 30°C and 80 to 100% relative humidity. Lesions developed on detached bracts within 72 h and were similar to those observed on the naturally infected plants. On tomato and chrysanthemum plants, pith necrosis and wilting symptoms were induced within 1 week of inoculation. Symptoms were not observed on control bracts and plants. Bacterial colonies were reisolated from bract lesions and stems with pith necrosis, but not from control plants, and the reisolates had the same LOPAT profile as the original isolates of P. viridiflava, thus fulfilling Kochs postulates. To our knowledge, this is the first report in the world of P. viridiflava causing a disease of artichoke bracts. References: (1) D. E. Goumas et al. Eur. J. Plant Pathol. 104:181, 1998. (2) Lelliott et al. J. Appl. Bacteriol. 29:470, 1966. (3) M. L. Saunier et al. Appl. Environ. Microbiol. 62:2360, 1996.

First Report of Bacterial Apical Necrosis of Mango Caused by Pseudomonas syringae pv. syringae in Greece

The mango, Mangifera indica L., native to India and Southeast Asia, is grown throughout the tropics and subtropics and marketed as fresh or processed fruit contributing greatly to those countries’ income. According to FAO database, from 2007 to 2012 the mango production in Greece reached the 438 tonnes, an increase of 338% indicating a strong economic potential for the cultivation. During spring of 2014 and 2015, symptoms similar to that of the bacterial apical necrosis disease caused by Pseudomonas syringae pv. syringae (Pss) were observed on commercial mango orchards planted with cv Irwin throughout two growing areas in Chania distinct, Crete, Greece. The disease was characterized by rapidly expanding necrotic lesions on leaves, buds, stems and floral panicles, whereas fruits were seemed to be un-affected. Lesions on leaves started as interveinal, angular, water soaked spots which may coalesce, becoming black and slightly raised (Figure 1). Isolations were made from affected buds, leaf petioles, and nec...

First Report of Root Rot and Vine Decline of Melon Caused by Monosporascus cannonballus in Greece

In June 2017, a severe vine decline of the netted melon (Cucumis melo L.) hybrids Global F1 and Lavigal was observed in a greenhouse of Messara valley, Heraklion, Crete, Greece. Diseased plants exhibited wilting, leaf chlorosis, yellowing and vine collapse. Root rot and necrosis, foot rot and to a lesser extent vascular tissue discoloration at the lower part of the stem were also observed. Numerous perithecia embedded in the root cortex, containing asci and ascospores typical of Monosporascus cannonballus Pollack & Uecker (1974) were observed. Disease incidence in the greenhouse was estimated at 100 and 78% for Global F1 and Lavigal, respectively. The fungus was isolated from the root lesions and the discolored vessels of the basal part of the stem, previously surface-sterilized with 1.5% sodium hypochlolrite for 1 min, on potato dextrose agar (PDA) amended with 0.5 g/l streptomycin sulfate. To obtain single hyphae isolates, hyphal tips were transferred onto new PDA plates and the growth rate of the fungu...