Massimo Delledonne Dipartimento di Biotecnologie
Università degli Studi di Verona

          Functional and Personal Genomics

Since 2006, my lab participated to the sequencing of the Vitis vinifera and Bifidobacterium dentium genomes, and performed an impressive number of microarray analyses of
genome wide gene expression. The development of high density microarrays led us to implement technologies for comparative genome hybridization, chromatin immunoprecipitation ("ChIP on chip") and sequence capture for targeted resequencing with 2nd generation sequencing technologies (NGS). As NGS has rapidly gained popularity for transcriptome analysis because of its ability to generate digital and quantitative information and to discover previously unknown genes, in 2008 we embraced gene expression analysis based on deep sequencing of the transcriptome (RNA-Seq). Since then, my lab has continued implementing and developing new wet-lab methodologies and bioinformatic pipelines for expression data analysis on genomic scales.

As the sequencing costs dropped, we begun involved in sequencing a number of bacterial, fungal and plant genomes. With surprise, we found that our experience on sequencing assembling and annotating the genome of "difficult" fungi and plants allowed us to smoothly approach human whole genome sequencing (WGS) and interpretation. Thanks to a lab composed by fantastic and enthusiastic people with different skills and very much different backgrounds (bioengineers, human and plant bioinformaticians, computer scientists, biotechnologists and geneticists), we are now taking full advantage of our understanding of
the complexity of different living organisms. It's amazing to see how much it can be learned when in the same lab people work on bacteria, plants and humans. A striking example is the comparison of genetic diversity in plant and humans: due to the young age (~60.000 years), to the fact that population has rapidly increased in the last 200 years or so (we were a 1 billion in 1800, 2 billion in 1930 and now increase a billion every 10-12 years) and to the continuous moving (and mating) of people all over the world, human genetic diversity is much limited compared to plants. Vitis vinifera for instance, like Arabidopsis and poplar, is a dicotyledonous plant that diverged from monocotyledons about 130–240 Myr ago. Being propagated asexually, each variety maintains a genetic diversity that goes beyond simple allele variation. The discovery that plant varieties/ecotypes can be caracterised by sets of proprietary genes and not only by a proprietary combination of different alleles of the same set of genes (like humans do) required a tremendous effort, as we had to adapt technologies mainly developed for human genomics that are based on resequencing and that therefore do not allow to characterise what is not present in the reference genome, forcing us to develop a "lateral thinking" approach.

The term "Lateral thinking" was coined in 1967 by Edward de Bono. Lateral thinking is solving problems through an indirect and creative approach, using reasoning that is not immediately obvious and involving ideas that may not be obtainable by using only traditional step-by-step logic. Lateral thinking deliberately distances itself from standard perceptions of creativity as either "vertical" logic (the classic method for problem solving: working out the solution step-by-step from the given data) or "horizontal" imagination (having a thousand ideas but being unconcerned with the detailed implementation of them).

Lateral thinking is now an integral component of our scientific approach.
Obviously, as we critically evaluate and discuss every single step of genome/transcriptome sequencing and data analysis, there is no room for "kit-persons" in the lab.

De-novo assembly of complex genomes is currently performed with Illumina paired ends and mate pairs (we produce MP up to 15 kb long), and are now testing optical maps (BioNano Genomics) for pseudomolecules reconstruction and scaffold anchoring. Genome annotation is based on MAKER and on the extensive use of directional RNA-Seq. We sequence whole human genomes (WGS) and exomes (WES) with Illumina (HiSeq 1000 and HiSeq X-Ten, the latter in outsourcing), an we perform targeted resequencing based on Agilent, Nimblegen and Illumina oligo capture, and amplicon sequencing (including Halopex technology). Human Genome analysis is based on BWA/GATK/MuTect/VarScan/Issac for alignment and variant calling. Variant interpretation is mainly based on SVS (GoldenHelix), IVA (Ingenuity) and KnoSOFT (Knome)

Comparison of the different technologies available and
testing of new emerging technologies for DNA sequencing and analysis is another integral part of our mission. For example, we have been deeply involved in the MinION Access Program to bring nanopore sequencing to maturity, and we are working side by side with Knome to build up a bioinformatics infrastructure that accelerates translational genomics initiatives and clinical NGS testing programs.

Whereas the lab is now deeply involved in genomic and transcriptomic projects with a special focus on the characterization of the "private" genome that we believe strongly contributes to make the difference among living organisms belonging to the same specie, I'm now particulary interested in human genome interpretation. I sequenced myself in 2011, and in 2013 I've joined the Understand Your Genome (UYG) program, a movement promoted by Illumina towards a better understanding of our DNA and its implications for healthcare
with the mission of accelerating a true era of precision medicine. I'm now serving UYG as Key Opinion Leader (KOL) to help catalyzing a cross-professional community united to responsibly yet urgently accelerate the adoption of genomics in healthcare.  

I'm a member of the Vigna-Vigne Consortium that sequenced the grape genome, of the International Cancer Genome Consortium and of the American Society of Human Genetics 

Nitric Oxide and Plant-Microbe Interactions

Nitric oxide (NO) is a highly reactive molecule that rapidly diffuses and permeates cell membranes. In animals, NO is implicated in a number of diverse physiological processes such as neurotransmission, vascular smooth muscle relaxation, and platelet inhibition. It may have beneficial effects, for example as a messenger in immune responses, but is also potentially toxic when the antioxidant system is weak and an excess of reactive oxygen intermediates (ROI) accumuates. During the last few years NO has been detected also in several plant species, and the increasing number of reports on its function in plants have implicated NO as an important effector of growth, development, and defense. The broad chemistry of NO involves an array of interrelated redox forms with different chemical reactivit
ies, and numerous potential targets of NO action exist in plants. NO signaling functions depend on its reactivity and ROI are key modulators of NO in triggering cell death, although through mechanisms different from those commonly observed in animals    

My laboratory specialises in the characterization of NO functions at cellular and molecular levels in plants. In collaboration with Chris Lamb we made pioneering work towards the discovery of NO function during the plant hypersensitive disease resistance response. We found that during the hypersensitive response plant cells accumulate NO, which co-operates with reactive oxygen species in the induction of hypersensitive cell death, and functions independently of such intermediates in the induction of defence related genes (Delledonne et al., 1998). We then demonstrated that the rates of production and dismutation of O2- generated during the oxidative burst play a crucial role in the modulation and integration of NO/H2O2 signalling in the hypersensitive response (Delledonne et al., 2001).

Due to the many possible mechanisms of NO action, a clear picture of its involvement in plant resistance to pathogens is far from being achieved. Our goal is now to characterize and modulate the signal transduction pathways leading to the hypersensitive disease resistance response. We are going in deep in the analysis of genes involved in the hypersensitive cell death and in the establishment of disease resistance whose expression is under control of NO. We built a NO fumigation platform that allow us to screen for mutants plants impaired in the activation of NO-triggered cell death, and we are now characterizing the first arabidopsis mutants that we have identified.
We are also focusing on the mechanisms regulating NO level in plant, and on the identification and characterization of signalling mechanisms that operate downstream of NO accumulation. In particular, we are analysing the occurrence of NO-dependent posttranslational modifications of proteins (S-nitrosylation and Tyr-nitration) to clarify their biological function and to understand their functional consequences in physiological and pathophysiological conditions.

The NO lab is now under the direct supervision of Diana Bellin (associate professor) and Elodie Vandelle (assistant professor)

Web pages of the 1st Plant Nitric Oxide Group Meeting, Verona 28-29 August 2006