Academic literature on the topic 'Settore BIO/04 - Fisiologia Vegetale'

One of the challenges that living organisms face is to respond promptly to genotoxic stress to avoid DNA damage. To this purpose, they developed complex DNA damage response (DDR) mechanisms. These mechanisms are highly conserved among organisms, including plants, and need to be finely regulated to take place properly. In this scenario, microRNAs are emerging as active players, thus attracting the attention of the research community. The involvement of miRNAs in DDR has been investigated prominently in human cells wherease studies on plants are still scarce. In addition, recently, miRNAs started to be envisioned as trans-kingdom molecules able to exert regulatory functions in evolutionary distant organisms. Particularly, attention is drawn to plant miRNAs ingested with the diet; evidence is accumulating on their ability to regulate genes in organisms other than the one in which they were synthesized, including humans and pathogens.In the present PhD thesis, different bioinformatics approaches have been developed aiming at identifying plant miRNAs along with their endogenous and cross-kingdom targets to pinpoint conserved pathways between evolutionary distant species. Alonside model organisms, the developed pipeline may find application on any species of interest to address species-specific cross-kingdom interactions or to performe large-scale investigations involving several plant/animal species. The emergence of DDR-related miRNAs in plants and humans constitutes fundamental informations obtained from these approaches.To experimentally investigate the involvement of plant miRNAs in the regulation of DDR-associated pathways, an ad hoc system was developed, using the model legume Medicago truncatula. Specific treatments with camptothecin (CPT) and/or NSC120686 (NSC) targeting compoments of DDR, namely topoisomerase I (Top1) and tyrosyl-DNA phosphodiesterase 1 (Tdp1), were used. These treatments, imposed to M. truncatula seeds for a 7-day time period, do not influence the germination process, but result in inhibition of seedling development, causing an increase in cell death and accumulation of DNA damage. To demonstrate that the imposed treatments had an effect on DDR, the expression of SOG1 (suppressor of gamma response 1) master-regulator was investigated by qRT-PCR. Importantly, a phylogenetic study demonstrated that M. truncatula possessed a small SOG1 gene family, composed by MtSOG1A and MtSOG1B genes. The expression of both genes was significantly enhanced in treatment-specific manner. Additionally, the espression of multiple genes playing important roles in different DNA repair pathways, cell cycle regulation and chromatin remodelling, were differentially expressed in a treatment-specific manner. Subsequently, specific miRNAs identifyed from the bioinformatics approach as targeting genes involved in DDR processes, were investigated along side their targets, thus providing a first step in their function validation.To investigate plant miRNAs trans-kingdom potential, additional studies were conducted using apple (M. domestica) since it can be eaten raw and hence, can be a better system for feeding trials. As a proof of concept, artificial miRNAs (amiRNAs) were delivered to human colorectal adenocarcinoma cells and the expression of these microRNAs and their in silico predicted targets were evaluated by qRT-PCR. Specifically, amiRNAs mimicking mdm-miR482a-3p and mdm-miR858 were transfected into HT-29 cell lines. After 72 h, amiRNAs were clearly detected inside the cells and the performed qRT-PCR analysis showed a significant downregulation of the IL4R (Interleukin 4 Receptor) gene, involved in promoting Th2 differentiation, suggesting the possibility of apple miRNAs to regulate the activity of human genes in vitro. Taken together, the results presented in the current PhD thesis demonstrate the involvement of plant miRNAs in DDR-associated processes as well as present evidence on the plant miRNAs trans-kingdom potential.
One of the challenges that living organisms face is to respond promptly to genotoxic stress to avoid DNA damage. To this purpose, they developed complex DNA damage response (DDR) mechanisms. These mechanisms are highly conserved among organisms, including plants, and need to be finely regulated to take place properly. In this scenario, microRNAs are emerging as active players, thus attracting the attention of the research community. The involvement of miRNAs in DDR has been investigated prominently in human cells wherease studies on plants are still scarce. In addition, recently, miRNAs started to be envisioned as trans-kingdom molecules able to exert regulatory functions in evolutionary distant organisms. Particularly, attention is drawn to plant miRNAs ingested with the diet; evidence is accumulating on their ability to regulate genes in organisms other than the one in which they were synthesized, including humans and pathogens.In the present PhD thesis, different bioinformatics approaches have been developed aiming at identifying plant miRNAs along with their endogenous and cross-kingdom targets to pinpoint conserved pathways between evolutionary distant species. Alonside model organisms, the developed pipeline may find application on any species of interest to address species-specific cross-kingdom interactions or to performe large-scale investigations involving several plant/animal species. The emergence of DDR-related miRNAs in plants and humans constitutes fundamental informations obtained from these approaches.To experimentally investigate the involvement of plant miRNAs in the regulation of DDR-associated pathways, an ad hoc system was developed, using the model legume Medicago truncatula. Specific treatments with camptothecin (CPT) and/or NSC120686 (NSC) targeting compoments of DDR, namely topoisomerase I (Top1) and tyrosyl-DNA phosphodiesterase 1 (Tdp1), were used. These treatments, imposed to M. truncatula seeds for a 7-day time period, do not influence the germination process, but result in inhibition of seedling development, causing an increase in cell death and accumulation of DNA damage. To demonstrate that the imposed treatments had an effect on DDR, the expression of SOG1 (suppressor of gamma response 1) master-regulator was investigated by qRT-PCR. Importantly, a phylogenetic study demonstrated that M. truncatula possessed a small SOG1 gene family, composed by MtSOG1A and MtSOG1B genes. The expression of both genes was significantly enhanced in treatment-specific manner. Additionally, the espression of multiple genes playing important roles in different DNA repair pathways, cell cycle regulation and chromatin remodelling, were differentially expressed in a treatment-specific manner. Subsequently, specific miRNAs identifyed from the bioinformatics approach as targeting genes involved in DDR processes, were investigated along side their targets, thus providing a first step in their function validation.To investigate plant miRNAs trans-kingdom potential, additional studies were conducted using apple (M. domestica) since it can be eaten raw and hence, can be a better system for feeding trials. As a proof of concept, artificial miRNAs (amiRNAs) were delivered to human colorectal adenocarcinoma cells and the expression of these microRNAs and their in silico predicted targets were evaluated by qRT-PCR. Specifically, amiRNAs mimicking mdm-miR482a-3p and mdm-miR858 were transfected into HT-29 cell lines. After 72 h, amiRNAs were clearly detected inside the cells and the performed qRT-PCR analysis showed a significant downregulation of the IL4R (Interleukin 4 Receptor) gene, involved in promoting Th2 differentiation, suggesting the possibility of apple miRNAs to regulate the activity of human genes in vitro. Taken together, the results presented in the current PhD thesis demonstrate the involvement of plant miRNAs in DDR-associated processes as well as present evidence on the plant miRNAs trans-kingdom potential.

Nei primi stadi dell’infezione, i microrganismi fitopatogeni producono un arsenale di enzimi che depolimerizzano in maniera ordinata e sequenziale i componenti della parete cellulare vegetale (CWDEs - Cell Wall Degrading Enzymes). Le poligalatturonasi (PG) sono tra i primi enzimi pectici ad essere prodotti e favoriscono la macerazione del tessuto vegetale rendendo accessibili a cellulasi ed emicellulasi gli altri componenti della parete. I carboidrati rilasciati dal processo degradativo della parete cellulare vengono utilizzati dal patogeno per il sostentamento e per la crescita, conferendo al processo di degradazione un ulteriore significato biologico oltre quello di distruzione fisica della parete. Le PGIP (PolyGalacturonase Inhibiting Proteins) presenti nella parete cellulare vegetale, sono delle glicoproteine in grado di inibire e/o modulare in maniera specifica l’attività delle PG. La formazione del complesso PG-PGIP rallenta la capacità delle PG di degradare l’omogalatturonano della parete cellulare, favorendo l’accumulo di oligogalatturonidi in grado di attivare le risposte di difesa della pianta. Questo lavoro vuole contribuire allo studio del ruolo delle PG in Phytophthora nicotianae e Phytophthora capsici, che sono ritenuti tra gli agenti patogeni più pericolosi per molte specie vegetali. Argomenti specifici che vengono affrontati in questa tesi riguardano: 1) l’identificazione di PG da alcune specie di oomiceti considerate tra le più pericolose per le piante (generi Phytophthora, Pythium ed Aphanomycetes); 2) l'analisi filogenetica delle famiglie geniche PG; 3) l’analisi di sequenze proteiche delle PG identificate allo scopo di rilevare domini e/o amminoacidi responsabili dell'interazione PG-PGIP; 4) la caratterizzazione delle PG da P. nicotianae e P. capsici; 5) la costruzione di mutanti di P. capsici per indagare il ruolo di PG nella patogenesi, utilizzando diversi approcci di genetica “reverse” I risultati ottenuti in questa tesi, potenziano l'ipotesi che la molteplicità delle PG può dare flessibilità al patogeno e ogni PG, con le proprie caratteristiche uniche, contribuisce alla performance del patogeno per colonizzare le piante con successo.
The plant cell wall is a structural barrier to pathogens, composed of a network of polysaccharides such as cellulose, hemicellulose and pectin. The majority of pathogenic microorganisms produce cell wall degrading enzymes (CWDEs) that are essential for the invasion process. Among the different CWDEs, polygalacturonases (PGs) play a critical role since their action on pectin makes other cell wall components more accessible to other CWDEs and causes tissue maceration. PGIPs (polygalacturonase-inhibiting proteins) are plant cell wall proteins that specifically modulate the activity of the PGs, and hamper the invasion process by limiting the host tissue colonization. The PG–PGIP interaction retards pectin hydrolysis and favors oligogalacturonide (OGs) accumulation and leading to plant defense activation. This work wants to contribute to study the role of the PGs in P. nicotianae and P. capsici, among the most dangerous pathogens for many plant species: Specific points of this thesis are: 1) Identification of the whole set of the PGs from well-known oomycetes, which present different lifestyles. 2) Comparison of large PG families found in the oomycetes species using phylogenetic analysis for tracking evolutionary relationships. 3) Analysis of amino acid sequences on identified PGs to detect domains and/or amino acids involved in PG-PGIP interaction. 4) Characterization of PGs from P. nicotianae and P. capsici. 5) Construction of P. capsici mutants for investigate the role of PGs in the pathogenesis, using different approach of reverse genetics. The results from this thesis enhances the hypothesis that the multiplicity of PGs may give flexibility to the pathogen, with each enzyme having its own unique properties to contribute to the performance of all the enzymes to successfully colonize plants.

As rich sources of flavonoids, carotenoids, vitamin C, folic acid and dietary fibre, fruits and vegetables can help prevent cardiovascular diseases and cancers (Amine et al. 2002). According to the world health report that was published by the WHO (2002), 2.7 million deaths per year can be attributed to low fruit and vegetable intake. Apple is the most common fruit crop grown in temperate regions (FAOSTAT 2012) and apples are affordable and widely available in most parts of the world. Apple and apple-derived products have been subject of many studies that have linked their intake to a beneficial effect on many diseases (Hyson 2011). Several studies have indicated a positive effect of phenolic compounds, which are abundantly present in apple, on preventing coronary heart disease and cancer (Hertog et al. 1993; Keli et al. 1996; Gerhauser 2008). The dietary fibres that are present in fruits like apple seem to decrease the risk for developing coronary heart disease, stroke, hypertension, diabetes, obesity and some gastrointestinal diseases (Anderson et al. 2009). Apples have shown to have high anti-oxidant activity and the intake of apple is associated with a reduced risk for heart disease, lung cancer, type 2 diabetes, and asthma (Boyer and Liu 2004), substantiating the beneficial effects of apple consumption on human health

Plants host complex fungal and bacterial communities on and inside various organs. These microbial populations could have beneficial and detrimental effects on their host. Despite the importance of plant bark as potential reservoir of plant pathogens and beneficial microorganisms for perennial crops, no information is available on the composition of bark microbiota under different climatic and agronomic conditions. Due to its agricultural and economic relevance, apple (Malus domestica) was selected as model system, in order to characterise the composition and functional properties of the bark microbiota. Most of the commercially relevant apple cultivars are susceptible to several destructive diseases and require intense use of fungicides. Scab-resistant apple cultivars allow the reduction of fungicide treatments and are compatible with low-input disease management strategies. However, fungicides applied to control apple scab can also control secondary pathogens and the reduction of fungicide applications on scab-resistant cultivars may cause the outbreak of emerging pathogens hosted on bark tissues, as for example the canker agent Diplodia seriata. Although the risk of emerging secondary pathogens is real under low-input disease management strategies, no information is available on the composition and dynamics of bark-associated microbial communities. The main aims of this work are i) to develop metabarcoding-based approaches for the precise study of barkassociated microbial communities and to investigate proportions of plant pathogens and beneficial microorganisms associated with young (one year-old shoots) and old barks (three/four-year-old shoots), ii) to identify impacts of environmental factors (orchard location and climatic conditions), host properties (plant species, cultivar and tissue age) and agricultural practices (integrated and low-input disease management) on the bark microbiota composition and iii) to evaluate fungicide impacts against a scarcely studied canker agent of apple under controlled conditions. A metabarcoding approach was optimized to characterise the composition of barkassociated microbial communities of two apple and two pear cultivars (Chapter II). Several potential plant pathogenic fungi (e.g. Alternaria spp., Diplodia spp., Gibberella spp., Peltaster spp., Penicillium spp., Phoma spp., Rosellinia spp., Stemphylium spp. and Taphrina spp.) and bacteria (e.g. Pseudomonas spp., Clavibacter spp., Curtobacterium spp., Nocardia spp.and Pantoea spp.) were found in presence of beneficial fungi (e.g. Arthrinium spp., Aureobasidium spp., Cryptococcus spp., Rhodotorula spp.,2 Saccharomyces spp. and Sporobolomyces spp.) and bacteria (e.g. Bacillus spp., Brevibacillus spp., Burkholderia spp., Deinococcus spp., Lactobacillus spp., Methylobacterium spp. and Sphingomonas spp.). The composition of bark communities was affected by the tissue age, plant species and cultivar and it strongly differed between young (one year-old shoots) and old barks (three/four-year-old shoots). The optimised protocol was then applied to characterize the bark-associated communities of the scabresistant apple cultivar Fujion under integrated and low-input disease management strategies in two different locations and consecutive seasons (Chapter III). In addition to the tissue age, orchard location and environmental conditions strongly affected fungal and bacterial richness and diversity of the bark communities. In particular, the orchard location affected the abundance of some potential plant pathogens, such as Alternaria spp., Cadophora spp., Diaporthe spp. Diplodia spp., Phoma spp. Curtobacterium spp., Pantoea spp. and Ralstonia spp. Moreover, the abundances of Cryptococcus, Leptosphaeria, Curtobacterium and Pseudomonas genera differed according to the disease management strategy, suggesting that it can shape the composition of plant-associated microbial communities. In particular, the low-input disease management strategy increased the abundances of sOTUs assigned to Alternaria, Cryptococcus, Rhodotorula, Curtobacterium, Methylobacterium, Rathayibacter and Rhizobacter, while the integrated disease management positively affected the abundances of Filobasidium, Erwinia and Pseudomonas. The functional characterization of apple secondary pathogens was then conducted on the canker agent D. seriata (Chapter IV). Diplodia seriata growth was inhibited by fungicides (captan, dithianon and fluazinam) in vitro although canker symptoms were not affected by dithianon on Fujion plants under greenhouse conditions. The overall work revealed a high complexity of the bark-associated microbial communities, highlighting bark as an overwintering site and reservoir of potential plant pathogens and beneficial microorganisms. Bark-associated fungal and bacterial communities were mainly affected by tissue age, orchard location, environmental conditions, disease management strategies, plant species and cultivars, indicating strong plasticity of the bark community to the environmental and host factors. However, cavities in the bark possibly protect microorganisms form perturbing factors and probably limit the efficacy of conventional fungicides against some secondary pathogens, such as D. seriata. Protocols optimised in this work can be further applied for the impact estimation of innovative agronomic practices (e.g. training systems and rain covers) on pathogenic and beneficial communities and for the precise assessment of D. seriata incidence under field conditions.

As a result of the constant demand to ensure food security and crop growth, there is an urgent need of a more sustainable and resilient agriculture. Chemical fertilisers and pesticides are widely used in conventional agriculture and they cause possible environmental impacts. Tomato (Solanum lycopersicum) is cultivated worldwide under field and greenhouse conditions, requires an extensive use of chemical fertilisers and is greatly affected by arthropod feeding damages. Microbial biofertilisers and non-microbial biostimulants can contribute to plant development. Among them, plant growth promoting endophytic bacteria can internally colonise plant tissues and promote plant growth. Likewise, humic acids (HA) are organic substances that improve soil structure and can facilitate plant nutrient uptake. However, scarce information is available on the synergistic effects of endophytic bacterial strains and HA on the bacteria-mediated plant growth promotion. Moreover, beneficial insects, including the generalist predators Macrolophus pygmaeus and Nesidiocoris tenuis, can be used in biological control for the management of crop arthropods. Despite the significance of insects as vectors of plant pathogens is unquestionable, the transmission of beneficial microbes by beneficial insects is unknown. The aims of this thesis were to get insight into the molecular basis of the interaction between endophytic bacterial strains and tomato plants in the presence of HA, in order to improve the understanding on the mechanism responsible for plant growth stimulation, and to investigate the possible use of beneficial insects as vectors to transmit selected endophytic bacterial strains for the further development of sustainable delivery strategies of bacterial biofertilisers. In order to understand the complementation effects and cellular pathways activated by endophytic bacterial strains and HA, three bacterial strains (namely Paraburkholderia phytofirmans PsJN, Pantoea agglomerans D7G and Enterobacter sp. 32A) that were able to promote tomato shoot length in the presence of HA were selected. Double labelling of oligonucleotide probes for fluorescent in situ hybridisation was used to study tomato colonisation by endophytic bacterial strains. In particular, the colonisation intensity of tomato root and stem among the tested strains were comparable in the control and HA condition. Moreover, transcriptomics approach was applied to study the molecular mechanisms activated in tomato shoots and roots in response to endophytic bacteria and HA. Tomato genes were modulated by endophytic bacterial strains mainly in roots, indicating major transcriptional regulations in 3 belowground compared to aboveground tissues. The majority of DEGs was modulated by more than two strains, involving protein metabolism, transcription, transport, signal transduction and defence, representing possible common pathways modulated in response to bacterial endophytes. Moreover, strain-specific tomato responses involved signal transduction, transcription, hormone metabolism, protein metabolism, secondary metabolism and defence processes, highlighting specific traits of the endophyte-tomato interaction. In particular, the presence of HA enhanced the activation of signal transduction, hormone metabolism, transcription, protein metabolism, transport, defence and growth-related processes in response to P. phytofirmans PsJN, P. agglomerans D7G and Enterobacter sp. 32A inoculation, in terms of number of modulated genes and fold change values, indicating additive effects of bacterial endophytes and HA in plant growth promotion mechanisms. In relation to the possibility to deliver endophytes by using insects, beneficial mirids of tomato (namely M. pygmaeus and N. tenuis) were tested as potential vectors to transmit P. phytofirmans PsJN and Enterobacter sp. 32A between tomato plants. Mirids feeding on seedinoculated tomato plants were able to acquire the beneficial bacterial strains. In particular, after contact with bacterial-inoculated plants, P. phytofirmans PsJN and Enterobacter sp. 32A were detected on the majority of mirid epicuticle and inside surface-sterilised insects. Moreover, both tested mirids transmitted the selected bacterial strains between tomato plants and P. phytofirmans PsJN and Enterobacter sp. 32A could be re-isolated from tomato shoots after mirid-mediated transmission. Our study demonstrated that P. phytofirmans PsJN and Enterobacter sp. 32A can move within tomato plants, from shoots to roots after mirid-mediated transmission, indicating that M. pygmaeus and N. tenuis can acquire non-pytopathogenic microbes and the polyphagous and mobile nature of mirids could facilitate the transmission of beneficial endophytes among crops. In summary, the overall work provide in-depth knowledge on the HA-dependent enhancement of growth-related processes stimulated by endophytic bacterial strains and demonstrates the potential of beneficial mirids in transmission of beneficial bacteria and paved the way for the further development of efficient HA- and mirid-mediated strategies for plant biofertilisation and beneficial bacteria delivery.

The proton pump interactor (Ppi) gene family of Arabidopsis thaliana: expression pattern of Ppi1 and characterisation of knockout mutants for Ppi1 and 2. Plant plasma membrane H+-ATPases (PM H+-ATPase) are essential for establishing a proton electrochemical gradient across the cell plasma membrane. Their regulation is poorly understood, except for the role of 14-3-3 proteins, which relieve autoinhibition from the C-terminal domain. A novel protein interacting with this domain was recently identified in Arabidopsis and named PPI1 (Proton Pump Interactor 1). PPI1 stimulates PM H+-ATPase activity in vitro. Here, we analyse the expression pattern of Ppi1 using b-glucuronidase as a reporter. Expression is strong in root and shoot vascular systems, particularly in meristematic and sink tissues, as well as in pollen, stigmas and siliques, but not in developing embryos. Removal of the first intron decreased GUS expression 45-fold. We also analysed the transcription of Ppi2, another gene in the family, and demonstrated that Ppi2 is expressed in seedlings, cultured cells and flowers. Insertional mutants for both Ppi1 and Ppi2 were isolated. Two different mutants of Ppi1 showed aberrant mRNAs and lacked any detectable protein and are therefore true knockouts. At the plant level, neither of the single mutants nor the double ppi1ppi2 mutant showed an altered phenotype in standard growth conditions under acid load or salt stress.